TW201011047A - Multispecific antibodies - Google Patents

Abstract

The invention provides multispecific antibodies and methods of making and using such antibodies.

Description

201011047 VI. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to multiplex specific antibodies, and methods of making and using the same. [Prior Art] The specific immunoglobulin polypeptide produced by the vertebrate immune system in response to challenge with foreign proteins, receptor proteins, cells or other antigenic foreign substances. An important part of this process is the production of antibodies that specifically bind to specific exogenous substances. The binding specificity of such polypeptides to specific antigens has been highly improved, and the multiple specificities that can be produced by individual vertebrates are significant in terms of their complexity and variability. Thousands of antigens can elicit. The reactions' reactions are almost exclusively directed to the specific antigen that elicits them. _ Specific antigen recognition is essential for antibodies to play a role in adaptive immune responses. In all vertebrates, the combination of heavy chain (HC) and light chain (lc) is conserved in the production of antibody lineages. However, there is a diversity of asymmetry in the two chains. The variable domain (Vh) & Lc of the HC _ variable domain (VL) contains significantly higher sequence diversity and more often supplies antigenic determinants. The role of LC in determining antigen specificity is indicated by the process known as receptor editing. The ongoing&recombinant recombination lines for editing B cell receptors are the primary mechanism for correcting self-reactive antibody precursors, which appear to constitute a large portion (about 75%) of the original lineage. Changes in the light chain have been demonstrated to eliminate unwanted binding specificity or multiple specificity. The specificity of antibodies and antibody fragments for a particular antigen can make the antibody an ideal therapeutic agent. Antibodies and antibody fragments can be used to target specific tissues (e.g., 142552.doc 201011047 tumors) and thereby minimize potential side effects in the non-specific age. Thus, there is a current and continuing need to identify and characterize therapeutic antibodies, particularly antibodies, fragments and derivatives thereof, suitable for the treatment of cancer and other proliferative disorders. SUMMARY OF THE INVENTION The present invention provides an isolated antibody comprising a high variable region (HVR) L1 sequence, wherein the HVR L1 sequence comprises the sequence NIAKTISGY (SEQ ID NO: 1), and wherein the antibody specifically binds to human epidermal growth factor Receptor 2 (HER2) and vascular endothelial growth factor (VEGF). In an embodiment, φ, the antibody further comprises: HVR-L2 comprising the sequence WGSFLY (SEQ ID ΝΟ: 2) and/or HVR-L3 comprising the sequence HYSSPP (SEQ ID ΝΟ: 3). In another embodiment, the antibody additionally comprises one, two or three _HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NIKDTY (SEQ ID ΝΟ: 4); (ii) the sequence RIYPTNGYTR ( HVR-H2 of SEQ ID NO: 5); and (iii) HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID ΝΟ: 6). In another embodiment, the antibody additionally contains one,

Two or three HVR sequences selected from the group consisting of (1) HVR-H1 containing the sequence NISGTY (SEQ ID NO: 7); (ii) HVR-H3 containing the sequence RIYPSEGYTR (SEQ ID NO: 9). In another aspect, the invention provides an isolated antibody comprising an HVR-L1 sequence, wherein the HVR-L1 sequence comprises the sequence XJXsXdsXeXvXsXaiSEQ ID NO: 83), wherein the guanidine is any amine group other than aspartic acid Acid, X3 is any amino acid other than proline, 142552.doc 201011047

X4 is any amino acid other than arginine and X5 is any amino acid other than serine, and wherein the antibody specifically binds to HER2 and VEGF. In one embodiment, the antibody comprising the sequence XJXsXiXsXeXTXsXpYCSEQ ID NO: 8 3) has aspartame, an alanine at X3, an amidic acid at X4, and sulphate at X5. , having a serine at X?, and/or having a glycine at X8, or any combination thereof. In various embodiments of this aspect of the invention, any of the HVR-L1 residues shown in Figure 57 having an F value greater than 1, 5 or 10 are preferably maintained as in bHl-44 or bHl- The residue found at the same position (SEQ ID ΝΟ: 1) of HVR-L1 of 81 is the same residue. In other embodiments, any of the HVR-L1 residues shown in Table 14 having an AAG value greater than 1 are preferably maintained at the same position as HVR-L1 of bH1-44 or bH1-81 (SEQ ID ΝΟ: 1) Residues found at the same residue. In one embodiment, the antibody comprises the HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 84). In one embodiment, the antibody further comprises: HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2) and/or HVR-L3 comprising the sequence HYSSPP (SEQ ID NO: 3). In another embodiment, the antibody further comprises one, two or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); (ii) the sequence RIYPTNGYTR (SEQ) ID NO: 5) HVR-H2; and (iii) HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO: 6). In another embodiment, the antibody additionally comprises one, two or three HVR sequences selected from the group consisting of (i) HVR-H1 comprising the sequence NISGTY (SEQ ID NO: 7); (ii) the sequence comprising RYPSEGYTR HVR-H2 of (SEQ ID NO: 8); and (iii) HVR-H3 having the sequence WVGVGFYAMD (SEQ ID NO: 9) containing 142552.doc 201011047. In another aspect, the invention provides an isolated antibody comprising an HVR-H2 sequence, wherein the HVR-H2 sequence comprises the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein X5 is any amine group other than threonine Acid and X6 is any amino acid other than aspartame, and wherein the antibody specifically binds to HER2 and VEGF. In another embodiment, the antibody comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID ΝΟ.84) has tyrosine at X8. In one embodiment, the antibody comprising the sequence RX2X3X4X5X6X7X8X9r (SEQ ID NO: 84) has leucine at 5 and/or glutamic acid at X6. In another embodiment of this aspect, the antibodies additionally comprise one, two or three HVR sequences which are selected from the group consisting of HVR-L1 containing the sequence NIAKTISGY (SEQ ID ΝΟ: 1). HVR-L2 containing the sequence WGSFLY (SEQ ID NO: 2) and/or HVR-L3 containing the sequence HYSSPP (SEQ ID NO: 3). In any of the embodiments described herein, the antibodies additionally comprise one or two HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4) and (ii) the sequence containing WGgdGFYAMD (SEQ. ID ΝΟ··6) H HVR-H3. In another embodiment, the antibodies additionally comprise one or two HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence msGTY (SEQ ID NO: 7) and (ii) the sequence containing WvgvGFYAMD (SEQ ID NO) :9) HVR-H3.

In various embodiments of this aspect of the invention, any of the HVR-H2 residues shown in Figure 57 having an f-value greater than 1, 5 or 1 均为 are preferably maintained as in bHl-44 or bHl. The residues found at the same positions of HVR-H2 of -81 (SEQ ID 142552.doc -6 - 201011047 NO: 8 and 5, respectively) are the same residues. In other embodiments, any of the HVR-H2 residues having an AAG value greater than 1 as shown in Table 14 are preferably maintained at the same position as HVR-H2 at bm-44 or bHl-81 (respectively The residues found at SEQ ID NO. 8 and 5) are the same residues. In a specific embodiment, the antibody comprises: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO: 1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2); and an HVR comprising HYSSPP (SEQ ID NO: 3) -L3 sequence; HVR-H1 sequence containing NIKDTY (SEQ ID NO: 4); HVR-H2 sequence containing RIYPTNGYTR (SEQ ID ΝΟ: 5); and HVR-H3 sequence containing WGGDGFYAMD (SEQ ID ΝΟ: 6), Or comprise: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID ΝΟ: 1); an HVR-L2 sequence comprising: WGSFLY (SEQ ID NO: 2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO: 3); The HVR-H1 sequence of NISGTY (SEQ ID NO: 7); the HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO: 8); and/or the HVR-H3 sequence containing WVGVGFYAMD (SEQ ID NO: 9). In another specific embodiment, the isolated antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which contains the sequence NIAKTISGY (SEQ ID ΝΟ: 1). WGSFLY (SEQ ID NO: 2); HYSSPP (SEQ ID NO: 3); NIKDTY (SEQ ID NO: 4); RIYPTNGYTR (SEQ ID NO: 5); and WGGDGFYAMD (SEQ ID NO: 6), and The antibody specifically binds to HER2 and VEGF. In another specific embodiment, the antibody comprises 1^11-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, 142552.doc 201011047 wherein each sequence contains the sequence NIAKTISGY (SEQ ID ΝΟ: 1); WGSFLY (SEQ ID NO: 2); HYSSPP (SEQ ID NO: 3); NISGTY (SEQ ID NO: 7); RIYPSEGYTR (SEQ ID NO: 8); and WVGVGFYAMD (SEQ ID NO: 9) And the antibody specifically binds to HER2 and VEGF. In various embodiments of any of the aspects described herein, the antibody binds human and murine VEGF with a Kd of 150 nM or greater and binds HER2 with a Kd of 7 nM or greater. In other embodiments, the antibody inhibits VEGF-induced cell proliferation and proliferation of HER2 expressing cells relative to a control. In a specific embodiment, the antibody binds to human and murine VEGF with a Kd of 36 nM or greater and binds to HER2 with a Kd of 1 nM or greater. In another embodiment, the antibody inhibits binding of VEGF to VEGFR2. In another aspect, the invention provides an isolated antibody that binds to human and murine VEGF with a Kd of 150 nM or greater and binds to HER2 with a Kd of 7 nM or greater, and wherein the antibody is relative to a control The substance inhibits VEGF-induced cell proliferation and proliferation of cells expressing HER2. In one embodiment, the antibody binds to human and murine VEGF with a Kd of 36 nM or greater and binds to HER2 with a Kd of 1 nM or greater. In yet another aspect, the invention provides an isolated antibody fragment that binds to human VEGF with a Kd of 58 nM or greater and KdS with HER2 of 6 nM or greater, and/or inhibits VEGF induction relative to a control Cell proliferation and proliferation of cells expressing HER2. In a specific embodiment, the antibody fragment binds human and murine VEGF with a Kd of 33 nM or greater and binds HER2 with a Kd of 〇.7 nM or greater. In another specific embodiment, the fragment 142552.doc 201011047 is a Fab fragment or a single chain variable fragment (scFv). In any of the above aspects, the antibody may be a monoclonal antibody. In another embodiment of all of the above aspects, the antibody can be an IgG antibody. In other embodiments of all of the above aspects, at least a portion of the framework sequences of the antibodies can be human consensus framework sequences. In another aspect, the invention provides a fragment of an antibody (any of the antibodies described herein). An example of an antibody fragment is a fragment that specifically binds to HER2 and VEGF and contains the HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1). In another embodiment, the antibody fragment additionally comprises one or two HVR sequences selected from the group consisting of (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); and (ii) the sequence containing HYSSPP ( HVR-L3 of SEQ ID - NO: 3). In another embodiment, the antibody fragment additionally comprises one, two or three HVR sequences selected from the group consisting of (i) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6). In another embodiment, the antibody fragment additionally comprises one, two or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NISGTY (SEQ ID NO: 7); (ii) the sequence comprising RIYPSEGYTR ( HVR-H2 of SEQ ID NO: 8); and (iii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In a particular embodiment, the antibody fragment comprises: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO: 1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2); comprising HYSSPP (SEQ ID NO: 3) HVR-L3 sequence; HVR-H1 sequence containing NIKDTY (SEQ ID NO: 4); HVR-H2 sequence containing 142552.doc 201011047 RIYPTNGYTR (SEQ ID NO: 5); and WGGDGFYAMD (SEQ ID NO: 6) HVR-H3 sequence, or comprising: HVR-L1 sequence containing NIAKTISGY (SEQ ID ΝΟ: 1); HVR-L2 sequence containing WGSFLY (SEQ ID NO: 2); HVR containing HYSSPP (SEQ ID NO: 3) -L3 sequence; HVR-H1 sequence containing NISGTY (SEQ ID NO: 7); HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO: 8); and HVR-H3 sequence containing WVGVGFYAMD (SEQ ID NO: 9). In one embodiment, the fragment is a Fab fragment or a single chain variable fragment (scFv). In other embodiments of all of the above aspects, at least a portion of the framework sequences of the antibodies can be human consensus framework sequences.

In other aspects, the invention provides polynucleotides encoding any of the antibodies or antibody fragments described herein, as well as a vector comprising such polynucleotides. In a specific embodiment, the encoded antibody comprises a HVR-L1 sequence comprising NIAKTISGY (SEQ ID ΝΟ: 1). Optionally or additionally, the polynucleotide encoding also comprises an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2) and/or an HVR-L3 sequence comprising HYSSPP (SEQ ID NO: 3) or Any combination of antibodies. In another aspect, the polynucleotide may additionally encode an antibody comprising one, two or three of the following sequences: an HVR-H1 sequence comprising NIKDTY (SEQ ID NO: 4); comprising RIYPTNGYTR (SEQ ID NO: 5) HVR-H2 sequence; and HVR-H3 sequence containing WGGDGFYAMD (SEQ ID NO: 6); or an antibody encoding one, two or three of the following sequences: containing NISGTY (SEQ ID NO: 7) HVR-H1; HVR-H2 containing RIYPSEGYTR (SEQ ID NO: 8); and/or HVR-H3 sequence containing WVGVGFYAMD (SEQ ID 142552. doc-10-201011047 NO: 9). In other aspects of the invention, the polynucleotide encodes an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7), an HVR-H2 sequence having RIYPSEGYTR (SEQ ID NO: 8) or has WVGVGFYAMD (SEQ ID) NO: 9) HVR-H3 sequence or any combination thereof. In other aspects, the invention provides an isolated polynucleoside acid encoding an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ·1), and optionally, the polynucleotide is further encoded One, two or three HVR sequences selected from the group consisting of (i) HVR-H1 comprising the sequence NIKDTY (SEQ ID ΝΟ: 4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID ΝΟ: 5); And (iii) HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID ΝΟ: 6). In other aspects, the invention provides an isolated polynucleotide encoding a sequence comprising: the HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1) and (i) the sequence containing WGSFLY (SEQ ID NO: 2) The HVR-L2 sequence or (ii) the HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO: 3) or both, and optionally, the polynucleotide encodes one, two or three An HVR sequence selected from the group consisting of (i) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and (iii) the sequence containing WGGDGFYAMD (SEQ ID NO: 6) HVR-H3. In another aspect, the invention provides an isolated polynucleotide encoding the sequence: HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1); HVR comprising the sequence NISGTY (SEQ ID NO: 7) -H1; containing 142552.doc -11 - 201011047 HVR-H2 having the sequence RIYPSEGYTR (SEQ ID NO: 8); and HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In still another aspect, the invention provides an isolated polynucleotide encoding the sequence: HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1); HVR comprising the sequence WGSFLY (SEQ ID NO: 2) -L2 sequence; HVR-L3 sequence containing the sequence HYSSPP (SEQ ID NO: 3); HVR-H1 containing the sequence NISGTY (SEQ ID NO: 7); HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO: 8); And HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In other aspects, the invention provides an isolated polynucleotide encoding a HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7), an HVR-H2 encoding the sequence RIYPSEGYTR (SEQ ID NO: 8) An isolated polynucleotide of the sequence and an isolated polynucleotide encoding the HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). In another aspect, the invention provides an isolated polynucleotide encoding a polypeptide comprising: an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7); comprising the sequence riypsEGYTR (SEQ ID) NO: 8) HVR-H2 sequence; and HVR-H3 sequence containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In another embodiment of the invention, the isolated polynucleotide encodes a HVR-L1 sequence comprising the sequence XJXsXAsXeXAsXgYCSEQ ID NO: 83), wherein Χι is any amino acid other than aspartic acid, X3 is Any amino acid other than proline, X4 is any amino acid other than arginine and X5 is any amino acid other than serine. In another embodiment of the invention 142552.doc -12-201011047, the polynucleotide encodes an HVR-L1 sequence comprising the sequence XiIX3X4X5X6X7X8X9Y (SEQ ID NO: 83) (where Xi is any amino acid other than Asp) , X3 is any amino acid other than proline, X4 is any amino acid other than arginine and X5 is any amino acid other than serine) and contains the sequence WGSFLY (SEQ ID NO: 2) The HVR-L2 sequence and/or the HVR-L3 sequence containing the sequence HYSSPP (SEQ ID NO: 3). In other embodiments of this aspect of the invention, the polynucleotide encoding contains sequences

An antibody of XilXsXAsXGXTXsXsYeEQIDNOJS) having aspartame at X!, having alanine at X3, an amine acid at X4, a sulphate at X5, a serine at X7, and/ Or have glycine at X8, or any combination thereof. In various embodiments of this aspect of the invention, any of the HVR-L1 residues shown in Figure 57 having an F value greater than 1, 5 or 10 are preferably maintained as in bHl-44 or bHl- The residue found at the same position (SEQ ID ΝΟ: 1) of HVR-L1 of 81 is the same residue. In other embodiments, any of the HVR-L1 residues shown in Table 14 having an AAG value greater than 1 are preferably maintained at the same position as HVR-L1 of bH1-44 or bH1-81 (SEQ ID ΝΟ: 1) Residues found at the same residue.

In another embodiment of the invention, the polynucleotide encodes an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein x5 is any amino acid other than threonine and x6 is in addition to the day Any amino acid other than benzalamine. In another aspect, the invention provides a polynucleotide encoding a sequence comprising the HVR-H1 sequence of the sequence NISGTY (SEQ ID NO: 7); comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID 142552.doc -13- 201011047 NO :85) the HVR-H2 sequence, wherein X5 is any amino acid other than threonine and X6 is any amino acid other than aspartame; and HVR containing the sequence WVGVGFYAMD (SEQ ID NO: 9) -H3 sequence. In another embodiment of the invention, the polynucleotide encodes an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 84), which has a serine at χ5 and a glutamic acid at x6. / or tartaric acid at χ8, or any combination thereof. In various embodiments of this aspect of the invention, any of the HVR-Η2 residues shown in Figure 57 having an F value greater than 1, 5 or 10 are preferably maintained as in bHl-44 or bHl- The residues found at the same positions of HVR-H2 at 81 (SEQ ID NOS: 8 and 5, respectively) are the same residues. In other embodiments, any of the HVR-H2 residues shown in Table 14 having a ΔΔG value greater than 1 are preferably maintained at the same position as HVR-H2 at bH1-44 or bH1-81 ( Residues identical to those found at SEQ ID NOS: 8 and 5), respectively. In other aspects, the invention provides an isolated polypeptide comprising the HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1), or an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1), An isolated polypeptide comprising the HVR-L2 sequence of the sequence WGSFLY (SEQ ID NO: 2) and/or the HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO: 3). In another aspect, the invention provides a polypeptide comprising: an HVR-L1 sequence comprising the sequence XJXaXAsXeXAsXgYGEQ ID NO: 83), wherein Χι is any amino acid other than aspartic acid, and X3 is a guanamine Any amino acid other than acid, x4 is any amino acid other than arginine and x5 is any amino acid other than serine. In another embodiment of this aspect 142552.doc •14· 201011047 Example 'The polypeptide contains the HVR-L1 sequence XJXgXAXsXeXyXsXsYCSEQ ID NO: 83) 'where X is any amino acid other than aspartic acid, χ3 In the case of any amino acid other than proline, χ4 is any amino acid other than arginine and X5 is any amino acid other than serine. Optionally, the polypeptide further comprises an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID ΝΟ: 2) and/or an HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID ΝΟ: 3). In a particular embodiment of any of the above aspects comprising a polypeptide comprising the sequence ID Ν〇: 83), the presence of asparagine at the oxime is at the presence of alanine at X3 and the presence of lysine at X4, The presence of threonine at the X5 is present at the sulphate, and/or the presence of glycine at & or any combination thereof. In various embodiments of this aspect of the invention, any of the HVR-L1 residues shown in Figure 57 having an f-value greater than 1, 5 or 1 均为 are preferably maintained as in bH1-44 or bHl. The residue found at the same position (SEQ ID NO: 1) of HVR-L1 of -81 is the same residue. In other embodiments, any of the HVR-L1 residues shown in Table 14 having a large value are preferably maintained at the same position as HVR-L1 of bH1-44 or bm-81 (SEQ ID 1 〇 :1) Residues found at the same residue. The invention also provides a polypeptide comprising: an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein X5 is any amino acid other than threonine and χ6 is any other than aspartame Amino acid. In another aspect of the invention, the polypeptide comprises the HVR-H2 sequence RX2x3x4x5x6x7X8X9R (SEq jd Ν〇:85) (wherein X5 is any amino acid other than threonine and χ6 is other than aspartame 142552.doc 15 201011047 Any amino acid), HVR-H1 sequence containing the sequence NISGTY (SEQ ID NO: 7) and HVR-H3 sequence containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In a different embodiment of the above aspect, the polypeptide comprising the HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 84) has a serine at χ5, glutamic acid at χ6, and/or at X8 Has tyrosine, or any combination thereof. In various embodiments of this aspect of the invention, any of the HVR-H2 residues shown in Figure 57 having an F value greater than 1, 5 or 10 are preferably maintained at bH 1 -44 or bH. Residues of residues found at the same positions of HVR-H2 of 1-8-1 (SEQ ID NOS: 8 and 5, respectively). In other embodiments, any of the HVR-H2 residues shown in Table 14 having an AAG value greater than 1 are preferably maintained at the same position as HVR-H2 at bHl-44 or bHl-81 (respectively The residues found at SEQ ID NOS: 8 and 5) are identical. The invention also provides a polypeptide comprising one, two or three of the following sequences: an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7), and an HVR-containing sequence RIYPSEGYTR (SEQ ID NO: 8) H2 sequence and/or HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9) or any combination thereof. In any of the above aspects, the isolated polypeptide may additionally comprise one, two or three of the following sequences: the HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1); comprising the sequence NIKDTY (SEQ ID NO: 4) HVR-H1; HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and/or HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6), or any combination thereof. 142552.doc -16- 201011047 In any of the above aspects, the isolated polypeptide may additionally comprise one, two or three of the following sequences: an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1); The HVR-L2 sequence of the sequence WGSFLY (SEQ ID NO: 2); the HVR-L3 sequence containing the sequence HYSSPP (SEQ ID NO: 3). In any of the above aspects, the isolated polypeptide may additionally comprise: HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and/or containing the sequence WGGDGFYAMD HVR-H3 (SEQ ID NO: 6), or any combination thereof.

In any of the above aspects, the isolated polypeptide may additionally comprise one, two or three HVR sequences selected from the group consisting of: HVR-H1 containing the sequence NISGTY (SEQ ID NO: 7); containing the sequence RIYPSEGYTR (SEQ ID) NO: 8) an HVR-H2 sequence; and/or HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NCh9), or any combination thereof. In other aspects, the invention provides an isolated polypeptide comprising: an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1) and (i) a HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2) a sequence or (ii) an HVR-L3 sequence comprising the sequence 歹|J HYSSPP (SEQ ID NO: 3) or both; and one, two or three HVR sequences selected from (i) containing the sequence NISGTY ( HVR-H1 of SEQ ID NO: 7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In other aspects, the invention provides an isolated polypeptide comprising: 142552.doc -17- 201011047 comprising the sequence 11乂11-1^1 of the sequence NIAKTISGY (SEQ ID 1 〇:1) and (0 containing the sequence HVR-L2 sequence of WGSFLY (SEQ ID NO: 2) or (ii) HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO: 3) or both; and one, two or three HVR sequences, Selected from: (i) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and/or (iii) containing the sequence WGGDGFYAMD (SEQ) ID NO: 6) HVR-H3 or any combination thereof. In other aspects, the invention provides an isolated polypeptide comprising the HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7), one comprising the sequence comprising RYPSEGYTR An isolated polypeptide of the HVR-H2 sequence of (SEQ ID NO: 8) and an isolated polypeptide comprising the HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID ΝΟ 9). In yet another aspect, the invention provides An isolated polypeptide comprising: an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7); an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); HVR-H3 sequence of WVGVGFYAMD (SEQ ID NO: 9). In one embodiment, the invention provides a vector comprising any of the above polynucleotides of the invention. In another aspect, the invention provides a Host cell of any of the vectors of the invention. In one embodiment, the host cell is prokaryotic. In another embodiment, the host cell is eukaryotic, such as a mammalian cell. In another aspect The invention provides a method of producing any of the above antibodies or antibody fragments, the method comprising culturing a host cell comprising a vector comprising a polynucleotide encoding the antibody 142552.doc.18-201011047 and recovering the antibody. Wherein the polynucleotide encodes an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1), and optionally, the polynucleotide encodes an HVR containing the sequence NIKDTY (SEQ ID NO: 4). H1, HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5) and HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6). In other embodiments, the polynucleotide encoding comprises the sequence NIAKTISGY (SEQ ID ΝΟ: 1) The HVR-L1 sequence, including The HVR-L2 sequence of the sequence WGSFLY (SEQ ID NO: 2) and the HVR-L3 sequence containing the sequence HYSSPP (SEQ ID ΝΟ: 3), and optionally, the polynucleotide encoding the sequence NIKDTY (SEQ HVR-H of ID ΝΟ: 4) contains HVR-H2 of the sequence RIYPTNGYTR (SEQ ID NO: 5) and HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO: 6). In another embodiment, the polynucleotide encodes an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1), a HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7), and a sequence containing the RYPSEGYTR (SEQ. ID NO: 8) HVR-H2 and HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In still another embodiment, the polynucleotide encodes an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1), an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO: 2), and a sequence containing HYSSPP (SEQ ID) NO: 3) HVR-L3 sequence, HVR-H1 containing sequence NISGTY (SEQ ID NO: 7), HVR-H2 containing sequence RIYPSEGYTR (SEQ ID NO: 8), and sequence containing WVGVGFYAMD (SEQ ID NO: 9) HVR-H3. In other embodiments, the polynucleotide encodes an HVR-H1 sequence comprising the sequence 142552.doc -19- 201011047 NISGTY (SEQ ID NO: 7), and an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8) Or the HVR-H3 sequence containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In still another embodiment, the polynucleotide encodes a polypeptide comprising: an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7); HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8) a sequence; and an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). In one embodiment, the host cell is prokaryotic, and in another embodiment, the host cell is eukaryotic, such as a mammalian cell. In another aspect, the invention provides a method of treating a tumor in an individual. The method comprises administering to the individual an antibody or antibody fragment described herein, wherein the administration is continued for a time and administered in an amount sufficient to treat or prevent the tumor in the individual. In one embodiment, the tumor is a colorectal tumor, breast cancer, lung cancer, renal cell carcinoma, glioma, glioblastoma or ovarian cancer. In another embodiment, the antibody comprises an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1) and specifically binds HER2 and VEGF. According to one embodiment, the antibody additionally comprises one or two HVR sequences selected from the group consisting of (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); and (ii) comprising the sequence HYSSPP (SEQ ID NO) :3) HVR-L3. In another embodiment, the antibody comprises one, two or three HVR sequences 歹 ij selected from (i) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); (ii) containing sequences HVR-H2 of RIYPTNGYTR (SEQ ID NO: 5); and (iii) HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO: 6). In another embodiment, the antibody contains one, two 142552.doc -20- 201011047

Or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence niSGTY (SEQ ID NO: 7); (ii) HVR-H2 comprising the sequence rIYPSeGYTR (SEQ ID NO: 8); and (iii) containing the sequence IDUR: 9) HVR-H3 ° In a particular embodiment, the antibody comprises: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID ΝΟ: 1), an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2), HVR-L3 sequence containing HYSSPP (SEQ ID NO: 3), HVR-H1 sequence containing NIKDTY (SEQ ID NO: 4), HVR-H2 sequence containing RIYPTNGYTR (SEQ ID NO: 5), and containing WGGDGFYAMD (SEQ ID NO: 6) The HVR-H3 sequence' and specifically binds to HER2 and VEGF. In another embodiment, the antibody comprises: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO: 1), an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2), and a HYSSPP (SEQ ID NO: 3) HVR-L3 sequence, HVR-H1 sequence containing NISGTY (SEQ ID NO: 7), HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO: 8), and HVR-H3 containing WVGVGFYAMD (SEQ ID NO: 9) Sequence, and specifically binds to HER2 and VEGF. In one embodiment, the method additionally comprises administering to the individual another anti-cancer therapy. In another embodiment, the additional anti-cancer therapy comprises another antibody, a chemotherapeutic agent, a cytotoxic agent, an anti-angiogenic agent, an immunosuppressant, a prodrug, a cytokine, a cytokine antagonist, a cytotoxic radiotherapy , corticosteroids, antiemetics, cancer vaccines, analgesics or growth inhibitors. In another embodiment, the additional anti-cancer therapy is administered prior to administration of the antibody 142552.doc • 21 - 201011047 or later. In another embodiment, the additional anti-cancer therapy is administered concurrently with the antibody. In another aspect, the invention provides a method of treating an autoimmune disease in an individual. The method comprises administering to the individual an antibody or antibody fragment described herein, wherein the administration is for a sustained period of time and administered in an amount sufficient to treat or prevent the autoimmune disease in the subject. In one embodiment, the antibody comprises the HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1) and specifically binds to HER2 and VEGF. According to one embodiment, the antibody comprises one or two HVR sequences selected from the group consisting of (1) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); and (ii) the sequence containing HYSSPP (SEQ ID NO: 3) HVR-L3. In another embodiment, the antibody comprises one, two or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); (ii) comprising the sequence RIYPTNGYTR (SEQ ID) NO: 5) of HVR-H2; and (iii) HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO: 6). In another embodiment, the antibody contains one,

Two or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NISGTY (SEQ ID NO: 7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID ΝΟ: 8); and (iii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In a particular embodiment, the antibody comprises: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO: 2), an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2), and an HVR-L3 sequence comprising HYSSPP (SEQ ID NO: 3) HVR-H1 sequence containing NIKDTY (SEQ ID NO: 4), HVR-H2 sequence containing RIYPTNGYTR (SEQ ID NO: 5), and HVR- containing WGGDGFYAMD (SEQ ID 142552.doc -22-201011047 NO: 6) H3 sequence, and specifically binds to HER2 and VEGF, or the antibody comprises: HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO: 1), HVR-L2 sequence containing WGSFLY (SEQ ID NO: 2), containing HYSSPP (SEQ ID NO: 3) HVR-L3 sequence, HVR-H1 sequence containing NISGTY (SEQ ID NO: 7), HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO: 8), and WVGVGFYAMD (SEQ ID NO: 9) HVR-H3 sequence, and specifically binds to HER2 and VEGF. In yet another aspect, the invention provides a method of treating a non-malignant disease involving abnormal activation of HER2 in an individual. The method comprises administering to the individual an antibody or antibody fragment described herein, wherein the time of administration and the amount administered are sufficient to treat or prevent the non-malignant disease in the individual. In one embodiment, the antibody comprises the HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1) and specifically binds to HER2 and VEGF. According to one embodiment, the antibody comprises one or two HVR sequences selected from the group consisting of (1) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); and (ii) comprising the sequence HYSSPP (SEQ ID NO: 3) HVR-L3. In another embodiment, the antibody further comprises one, two or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); (ii) the sequence RIYPTNGYTR (SEQ) ID NO: 5) HVR-H2; and (iii) HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO: 6). In another embodiment, the antibody comprises one, two or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NISGTY (SEQ ID NO: 7); (ii) comprising the sequence RIYPSEGYTR (SEQ ID) NO: 8) of HVR-H2; and (iii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In a specific example 142552.doc -23-201011047, the antibody comprises: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID ΝΟ: 1), an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2), and a HYSSPP (including HVR-L3 sequence of SEQ ID NO: 3), HVR-H1 sequence containing NIKDTY (SEQ ID NO: 4), HVR-H2 sequence containing RIYPTNGYTR (SEQ ID NO: 5), and WGGDGFYAMD (SEQ ID NO: 6) HVR-H3 sequence, and specifically binds to HER2 and VEGF, or the antibody comprises: HVR-L1 sequence comprising NIAKTISGY (SEQ ID ΝΟ: 1), HVR-L2 sequence containing WGSFLY (SEQ ID NO: 2) The HVR-L3 sequence containing HYSSPP (SEQ ID NO: 3), the HVR-H1 sequence containing NISGTY (SEQ ID NO: 7), the HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO: 8), and WVGVGFYAMD The HVR-H3 sequence of (SEQ ID NO: 9), and specifically binds to HER2 and VEGF. Other aspects of the invention provide the use of the antibodies and anti-sputum fragments described herein for the treatment of a tumor, autoimmune disease or non-malignant disease involving abnormal activation of HER2 in an individual, and for the manufacture of HER2 for the treatment of an individual Use of an abnormally activated tumor, an autoimmune disease, or a drug for a non-malignant disease. In one embodiment of such use, the antibody comprises an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1) and specifically binds to HER2 and VEGF. According to one embodiment, the antibody additionally comprises one or two HVR sequences selected from the group consisting of (1) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); and (ii) the sequence comprising HYSSPP (SEQ ID NO: 3) )

HVR-L3. In another embodiment, the antibody comprises one, two or three HVR sequences selected from the group consisting of: (1) HVR-H1 comprising the sequence NIKDTY (SEQ ID 142552.doc • 24-201011047 NO: 4); HVR-H2 comprising the sequence riyptNGYTR (SEQ ID NO: 5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6). In another embodiment, the antibody comprises one, two or three HVR sequences selected from the group consisting of (1) HVR-H1 comprising the sequence NISGTY (SEQ ID NO: 7); (ii) comprising the sequence riypsEGYTR (SEQ ID) NO: 8) HVR-H2; and (iii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID ΝΟ··9). In a particular embodiment, the antibody comprises: an HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO: 1), an HVR-L2 sequence comprising WGSFLY (SEQ ID NO: 2), and a HYSSPP (SEQ ID NO: 3) HVR-L3 sequence, HVR-H1 sequence containing NIKDTY (SEQ ID NO: 4), HVR-H2 sequence containing RIYPTNGYTR (SEQ ID NO: 5), and HVR containing WGGDGFYAMD (SEQ ID NO: 6) H3 sequence, and specifically binds to HER2 and VEGF, or the antibody comprises: HVR-L1 sequence containing NIAKTISGY (SEQ ID ΝΟ: 1), HVR-L2 sequence containing WGSFLY (SEQ ID NO: 2), containing HYSSPP ( The HVR-L3 sequence of SEQ ID NO: 3), the HVR-H1 sequence containing NISGTY (SEQ ID NO: 7), the HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO: 8), and WVGVGFYAMD (SEQ ID NO: 9) The HVR-H3 sequence, and specifically binds to HER2 and VEGF. In one embodiment of the method of treating a tumor, autoimmune disease or non-malignant disease involving aberrant activation of HER2 as described herein, the individual is a human. In addition, kits, compositions, and articles of manufacture comprising the antibodies and antibody fragments described herein are contemplated. 142552.doc • 25- 201011047 [Embodiment] The present invention provides methods for producing multispecific antibodies and antibody fragments, and antibodies recognized by the methods and uses thereof. In general, the methods of the invention comprise diversifying the light chain variable domain or heavy chain variable domain of an antibody to produce a variant that is stable in the library. Diversified antibodies that specifically bind to two epitopes were subsequently selected from this library and further characterized. Exemplary antibodies recognized using the methods of the present invention include antibodies that bind to both HER2 (human epidermal growth factor receptor 2) and VEGF (vascular endothelial growth factor). In particular, the information described herein (e.g., in the Examples below) demonstrates that mutations in the light chain complementarity determining regions (CDRs) of the HER2 antibody confer dual binding ability to the unrelated protein antigen and HER2. A bispecific high affinity HER2/VEGF antibody has been extensively characterized. In addition, the crystal structure of this bispecific Fab complexed with HER2 and VEGF was displayed and the energy contribution of the mutation-induced Fab residue was evaluated. The binding sites for the two antigens overlap extensively; most of the CDR residues that contact HER2 also occlude VEGF. However, in terms of energy, the residues of the heavy chain dominate the HER2 specificity, while the light chain dominates the VEGF specificity. The HER2/VEGF bispecific antibody inhibits HER2 and VEGF-mediated cell proliferation in vitro and in vivo. These results indicate that altering the sequence of the light chain variable domain of an antibody produces antibodies with bispecificity and function. For example, bHl-44 and bHl-81 have the potential to target two tumor progression mechanisms: tumor cell proliferation mediated by HER2 and tumor angiogenesis mediated by VEGF. Co-targeting two antigens with a single antibody is an alternative to combination therapy 142552.doc -26- 201011047. ι.Definition

The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (vL), wherein the VHVL unit has multiple epitope specificity (ie, , capable of binding to two different epitopes on one biomolecule or each antigen on a different biomolecule). Such multispecific antibodies include, but are not limited to, full length antibodies, antibodies having two or more VL&VH domains, such as Fab, Fv, dsFv, scFv, bifunctional antibodies, bispecific bifunctional antibodies, and An antibody fragment of such an antibody, a covalently or non-covalently linked antibody fragment. "Multiple epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Monospecific" refers to the ability to bind only one epitope. According to one embodiment, the multispecific antibody binds to each of 5 μΜ to 0.001 pM, 3 μΜ to 0.001 ρΜ, 1 μΜ to 0.001 ρΜ, 〇·5 μΜ to 0.001 ρΜ or 0.1 μΜ to 0.001 ρΜ. The IgGl form of the epitope. A basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains and two identical heavy (H) chains (IgM antibodies are composed of 5 basic heterotetramer units and the other is referred to as J-chain The polypeptide consists of, and thus contains, one antigen binding site' and the secreted IgA antibody can be polymerized to form a multivalent aggregate comprising 2 to 5 basic 4-chain units and a J chain. In the case of 1 § ,, the 4-chain unit is typically about 15 〇, dalton. Each L chain is linked to the oxime chain by a covalent disulfide bond, and the two oxime chains are linked to each other via one or more disulfide bonds depending on the oxime chain isotype. Each Η chain and L chain also has a regular interval 142552.doc -27- 201011047. Each H chain has a variable domain (Vh) at the end of the ^, followed by three constant domains (CH) for each of the α & gamma chains and four CH domains for the μ and ε isotypes. Each l chain has a variable domain (VL) at the ν terminus, and is followed by a constant domain (Cl) at its other end. The vl system is aligned with vH and (the first and the first of the heavy chain are 'I· Domain (CH1) alignment. The specific amino acid residue forms an interface between the light chain and the heavy chain variable domain. The pairing of Vh and VL together form a single antigen binding site. And characteristics, see for example Basic and Clinical Immunology, 8th edition, Daniel P.

Stites, Abba I. Terr and Tristram G. Parslow (ed.), Appleton & ❿ Lange, Norwalk, CT, 1994, pp. 71 and 6. The amino acid sequence of the L chain of any vertebrate species based on its constant domain can be assigned to one of two distinct types (referred to as < and λ). Immunoglobulins may be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of their heavy chain. There are five types of immunoglobulins: IgA, igD,

IgE, IgG, and IgM' have heavy bonds named α, δ, γ, ε, and μ, respectively, and the α class is based on relatively small differences in cH sequences and functions.

Further divided into subclasses, for example, humans exhibit the following subclasses IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The term "variable" refers to the fact that the sequences of certain segments of the variable domain vary widely between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 11 amino acid spans of the variable domain. In fact, the V region consists of a relatively invariant sequence of 15 to 30 amino acids called the framework regions (FR), which are called 9 to 12 amino acids each. The hypervariable zone is separated by a shorter zone of extreme variation of H2552.doc -28- 201011047. The variable domains of the native heavy and light chains each comprise four FRs that are predominantly joined by a beta-sheet configuration, joined by three hypervariable regions, which form a loop connecting the beta-sheet structure, and in some cases Forming a portion of the beta sheet structure. The hypervariable regions in each chain are held together in close proximity by the FR and, together with the hypervariable regions of the other chain, promote the formation of antigen binding sites for antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, section 5). Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Strange localization is not directly related to binding antibodies to antigens, but exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" as used herein, refers to an amino acid residue of an antibody responsible for antigen binding. The hypervariable region typically contains an amino acid residue of the "complementarity determining region" or "CDR" (eg, at about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL. Surrounding, and in VH around about residues 26-35 (Η1), 50-65 (H2), and 95-102 (H3) (in one embodiment, H1 is around about residues 1 1-35) ); Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public

Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or their residues of the "hypervariable loop" (eg, residues 26-32 (Ll) in VL '50-52 (L2) And 91-96 (L3), and in VH are 26-32 (H1), 53-55 (H2) and 96-101 (H3); Chothia and Lesk, J. Mol· Biol. 196:901-917 (1987)). "Framework regions" (FR) are those variable domain residues other than CDR residues. Each variable domain typically has four 142552.doc -29- 201011047 FRs identified as FR1, FR2, FR3, and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are located at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain The FR residues are located at about residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the CDR comprises a hypervariable cyclic amino acid residue, the light chain FR residue is located in the light chain at about residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and 97 -107 (LCFR4), and the heavy chain FR residues are located at about residues 1-25 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues. ). In certain instances, when the CDR comprises both an amino acid such as the CDR of Kabat and an amino acid of a hypervariable ring, the FR residue will be adjusted accordingly. For example, when CDRH1 comprises the amino acid H26-H35, the heavy chain FR1 residue is at positions 1-25 and the FR2 residue is at positions 36-49. "Human common framework" is a framework representing the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the subpopulation of sequences is a subgroup such as in Kabat. In one embodiment, for VL, the subpopulation is a subgroup κ I as in Kabat. In one embodiment, for VH, the subpopulation is a subgroup such as Kabat. The term "monoclonal antibody" as used herein refers to an antibody from a population of substantially homogeneous antibodies', ie, possible variants of the individual antibodies that make up the group, except that may be produced during the production of a monoclonal antibody and are generally present in minor amounts. It is substantially identical except for the same and binds to the same epitope. The monoclonal antibody generally comprises an antibody comprising a variable region that binds to a target, wherein the anti-system is obtained by selecting an antibody from a plurality of antibodies by 142552.doc • 30- 201011047. For example, the selection method may select a unique pure line from a plurality of pure lines (such as a fusion tumor pure line, a phage pure line, or a collection of recombinant DNA pure lines). It will be appreciated that selected antibodies may be further modified, for example, to improve affinity for the target, to humanize the antibody, to improve its production in cell culture, to reduce its in vivo immunogenicity, to generate multiple specific antibodies, etc. And it should be understood that the antibody comprising the modified variable region sequence is also a monoclonal antibody of the invention. In addition to its specificity, monoclonal antibody preparations are advantageous in that they are generally not contaminated by other immunoglobulins. The modifier "single plant" means the characteristics of an antibody obtained from a population of substantially homologous antibodies and is not intended to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques, including fusion knob methods (e.g., Kohler et al.,

Nature, 256:495 (1975) ; Harlow et al., Antibodies: A

Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., Monoclonal

Antibodies and T-cell Hybridomas 563-681 (Elsevier, NY, 1981), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), bacteriophage presentation technology (see, for example, Clackson et al, Nature, 352: 624-628 (1991); Marks et al, J. Mol Biol, 222: 581-597 (1991); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee Et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al. Immunol. Methods 284(1-2): 119-132 (2004)) and for the production of human antibodies or from humans having human immunoglobulin sequences. 142552.doc • 31 - 201011047 Immunoglobulin loci or genes Techniques for human or antibody-like antibodies to some or all of the animals (see, for example, W098/24893, WO/9634096, WO/9633735, and WO/91 10741, Jakobovits #A, Proc. Natl. Acad. Sci. USA, 90:2551 ( 1993); Jakobovits et al, Nature, 362: 255-258 (1993); Bruggemann et al, Year in Immuno., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591, No. 669 (owned by GenPharm), No. 5,545,807, WO 97/17852, U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016, and Marks et al., Bio /Technology, 10: 779-783 (1992); Lonberg et al, Nature, 368: 856-859 (1994);

Morrison, Nature, 368: 812-813 (1994); Fishwild et al.

Nature Biotechnology, 14:845-851 (1996) » Neuberger, Nature

Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern.

Rev. Immunol 13:65-93 (1995)) o A "intact" antibody is an antibody comprising an antigen binding site and CL and at least a heavy chain constant domain (CH1, CH2 and CH3). The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions. An "antibody fragment" comprises a portion of an intact antibody, preferably an antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; bifunctional antibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10): 105 7-1062 (1995)); single-chain antibody molecule; and 142552.doc-32-201011047 multispecific antibody formed from antibody fragment. The expression "linear antibody" generally refers to the antibody described in Zapata et al., Protein Eng., 8(10): 1057-1062 (1995). Briefly, the antibodies comprise a pair of tandem Fd segments (Vh-Ch1-Vh-Ch1) which together with the complementary light chain polypeptide form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. Papain-digested antibodies produce two identical antigen-binding fragments, termed "Fab" fragments and residual "Fc" fragments, which reflect the ability to crystallize readily. The Fab fragment consists of the entire L chain as well as the variable region (vH) of the Η chain and the first constant domain (CH1) of the heavy chain. Pepsin treatment of the antibody produces a single large F(ab')2 fragment that roughly corresponds to the two disulfide-linked Fab fragments with bivalent antigen binding activity and is still capable of cross-linking the antigen. • The Fab' fragment differs from the Fab fragment by having an extravagant residue at the carboxy terminus of the CH1 domain' comprising one or more semi-proline from the antibody chimeric region. Fab'-SH is herein a nomenclature for Fab' with a free thiol group for a constant domain cysteine residue. The F(ab')2 antibody fragment was originally generated as a Fabi fragment pair with hinged cysteine between the fragments. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises a carboxy terminal portion of two η strands held together by a disulfide bond. The effector function of an antibody is determined by the sequence in the region; this region is also the portion recognized by the Fc receptor (FcR) found on a particular type of cell.

Fv" consists of a dimer of a heavy chain variable region that is tightly non-covalently associated with a variable region of a light chain. From the folding of the two domains, six 142552.doc •33·201011047 hypervariable loops (three loops each from the η and L chains), which contribute to the antigen binding of the amino acid residues and confer antigen to the antibody Binding specificity. However, even if a single variable domain (or one half of an Fv comprising only three antigen-specific Cdrs) has the ability to recognize and bind antigen, the affinity is often lower than the entire binding site. A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising a VH and VL antibody domain linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the sFv is capable of forming the desired structure for antigen binding. For a review of sFv, see

Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, ed. R〇senburg and Moore, Springer-Verlag, New York pp. 269-315 (1994); Borrebaeck 1995. The term "bifunctional antibody" refers to a small antibody fragment prepared by constructing an sFv fragment (see the previous paragraph) having a short linker (about 5 to 10 residues) between the VH and VL domains such that the V domain is achieved. Inter-strand pairing rather than intra-pair pairing produces a bivalent fragment, ie a fragment with two antigen binding sites. A bispecific bifunctional antibody is a heterodimer of two "crossover" sFv fragments in which the VH and VL domains of two antibodies are present on different polypeptide chains. Bifunctional antibodies are described more fully in, for example, EP 404,097, WO 93/11161 and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing minimal sequence derived from a non-human antibody. To a large extent, humanized antibodies are non-human species (such as mouse, rat, rabbit or non-142552.doc-34-) in which the residues of the receptor hypervariable region have the desired antibody specificity, affinity and ability. 201011047 Human primate (receptor antibody) in which the residues of the hypervariable region of the human primate (donor antibody) are replaced. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody potency. In general, a humanized antibody will comprise substantially all of at least one (and generally two) variable domains, wherein all or substantially all of the hypervariable loops correspond to a hypervariable loop of a non-human immunoglobulin and all or In general, all FRs are ©® FR of human immunoglobulin sequences. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al, Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); &Presta, Curr.Op· Struct. Biol. 2:593-596 (1992). As used herein, "codon set" refers to a set of different nucleotide triplet sequences encoding the desired amino acid. For example, a set of oligonucleotides can be synthesized by solid phase synthesis, including all possible combinations of nucleotide triplets provided by the codon set and which will encode the sequence of the desired amino acid group. The standard form of codon nomenclature is the standard form of the IUB code, which is known in the art and is described herein. The codon set is generally represented by italics in three uppercase letters, such as NNK, NNS, XYZ, DVK, and the like (eg, the NNK codon refers to the 1 and 2 positions of the codon, N=A/T/ G/C and K=G/T at the 3 position in the equimolar ratio to encode all 20 natural amino acids). Thus, as used herein, "non-random codon set" refers to a code that selects (preferably completely) a selected amino acid that meets the criteria for amino acid selection 142552.doc-35-201011047 as described herein. Subgroup. The synthesis of oligonucleotides having a degeneracy of the selected nucleotide acid at a particular position is well known in the art, such as the TRIM method (Knappek et al, J. Mol. Biol. 296: 57-86, 1999); Garrard and Henner, Gene 128: 103, 1993. Such sets of nucleotides with a particular codon set can be synthesized using commercially available nucleic acid synthesizers (eg, 'commercially available from Applied Biosystems, Foster City, CA) or commercially available (eg, 'obtained from Life Technologies, Rockville , MD) ° Therefore, a group of nucleotides having a particular codon set synthesized will generally comprise a plurality of oligonucleotides having different sequences, which are established by codon sets within the total sequence. Oligonucleotides as used in accordance with the invention have sequences which allow hybridization to a variable domain nucleic acid template and may, but need not, include restriction enzyme sites suitable for, for example, selection purposes. The antibody of the "antibody" of the antigen of interest of the present invention binds the antigen with sufficient affinity to make the antibody suitable as a diagnostic and/or therapeutic agent in the target protein or the cell or tissue expressing the antigen and does not significantly cross-react with other proteins.

Antibody. In such embodiments, the antibody binds to the "non-target" protein to a lesser extent than the antibody binds to its specific target protein as determined by fluorescence activated cell sorting (facs) analysis or radioimmunoprecipitation (RIA) 4 ELISA. About 10/. . With respect to the binding of an antibody to a target molecule, the term "specifically binds/or specifically binds to an epitope on a particular polymorphic or specific polymorphic stem" or "marks a specific polypeptide or a specific polypeptide" "Antigenic epitope specific" means a population that is different from the non-specific interaction = for example, for _44 or bH1_81, non-specific interactions to bovine serum albumin, road protein, fetal Bovine serum or neutral bond avidin 142552.doc -36 - 201011047 (neuravidin)). For example, specific binding can be detected by determining the binding of a molecule as compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to a standard (e.g., 'excess unlabeled handles') to determine specific binding. In this case, if the binding of the labeled label to the probe is competitively inhibited by excess unlabeled target, then the specific binding is indicated. The term "specifically binds to a specific polypeptide or an epitope on a particular polypeptide" or "specifically binds to an epitope on a particular polypeptide or a particular polypeptide" or "specific polypeptide or specificity" as used herein. The epitope specificity on the peptidomimetic target can be, for example, at least about 200 nM, or at least about 150 nM, or at least about 1 〇〇 nM, or at least about 60 nM, or at least about 5 Å from the target. nM, or at least about 40 nM, or at least about 30 nM, or at least about 2 nM, or at least about nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 A molecule of nM, or at least about 4 μm or larger, is shown. In the embodiment of item f, the term "specifically binds" refers to a combination of a molecule that binds to a specific polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "binding affinity" refers to the inherent 尨 σ affinity that reflects the 丨·丨 interaction between a member (e. g., an antibody and an antigen). The affinity of the molecule X for its conjugate γ is usually represented by the dissociation constant (Kd). Desirably, Kd is about 2〇〇ηΜ, ι5〇142552.doc .37- 201011047 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM '20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or stronger. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies typically bind antigen slowly and tend to be readily dissociated, while high-affinity antibodies typically bind antigen faster and tend to remain bound for a longer period of time. A variety of methods for measuring binding affinity are known in the art, and any of these methods can be used to achieve the objectives of the present invention. In one embodiment, using BIAcoreTM-2000 iBIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ), by using a surface electrical resonance assay, at about 1 unit of reaction unit at 25 ° C (RU The fixed antigen CM5 wafer is used to measure the "Kd" or "Kd value" according to the present invention. Briefly, N-ethyl-N'-(3-diamidinopropyl)carbodiimide hydrochloride (EDC) and N-hydroxybutylimine (NHS) were used according to the supplier's instructions. The carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc.) was activated. The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μL/min to obtain about 10 reaction units (RU) of the coupling protein. After the antigen was injected, 1 Μ ethanolamine was injected to block the unreacted group. For kinetic measurements, two-fold serial dilutions (e.g., 0.78 nM to 500 nM) of Fab were injected at 25 ° C at a flow rate of about 25 μΐ/min in PBS with 0.05% Tween 20 (PBST). The association rate (Ιη) and the dissociation rate (kQff) were calculated by simultaneously fitting the association and dissociation sensor profiles by using one of the simpler Langmuir binding models (BIAcore evaluation software version 3.2). Calculate the balance based on the ratio 142552.doc -38- 201011047 Dissociation constant (Kd). See, for example, Chen, Y. et al., (1999) j. Biol. 293:865-881. If the combined rate of the above surface plasma resonance is more than 106 M·1 S·1, it can be stopped in a spectrometer (such as equipment).

A spectrophotometer (Aviv Instruments or 8000 series SLM-Aminco spectrophotometer (ThermoSpectr〇nie) with red light) ~ measured in the presence of increasing concentrations of antigen, used at 25 ° C Determination of the increase or decrease in the fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) by fluorescence quenching technique (excitation = 295 nm; emission = 340 nm '16 nm band pass) Association rate. According to the invention, the "association rate" or "kcn" can also be used at 25 ° C using BIAcoreTM-2000 or BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) in about 10 reaction units (RU). The immobilized antigen CM5 wafer was measured using the same surface plasma resonance technique described above. Briefly, N-ethyl-N'-(3-diamidinopropyl)carbodiimide hydrochloride (EDC) and N-hydroxybutylimine (NHS) were used according to the supplier's instructions. The carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc.) was activated. The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), and then injected at a flow rate of 5 μl/min to obtain about 1 unit of reaction unit (RU) of the coupled protein. After the antigen was injected, 1 Μ ethanolamine was injected to block the unreacted group. For kinetic measurements, two-fold serial dilutions of Fab (eg ''·78 nM to 500) were injected at 25 ° C at a flow rate of approximately 25 μΐ/min in PBS (PBST) with 0.05% Tween 2〇. nM). Calculate the association rate and dissociation by using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2) by simultaneously fitting the association and dissociation sensor maps. 142552.doc -39- 201011047 Rate (kQff) . The equilibrium dissociation constant (Kd) was calculated from the ratio kcff/kon. See, for example, Chen, Y. et al., (1999) J. Mol. Biol. 293:865-881. However, 'if the association rate obtained by the above surface plasma resonance test exceeds 106 Μ 1 S 1 ', it may preferably be in a spectrometer such as a spectrophotometer equipped with a flow stop device (Aviv Instruments) or with a stirring phototube. 8000 Series SLM-Aminco Spectrophotometer (ThermoSpectr〇nic)) measured in the presence of increasing concentrations of antigens by measuring 20 nM anti-antibody (Fab format) in PBS (pH 7.2) at 25 °C Fluorescence quenching techniques for increasing or decreasing the intensity of the fluorescence emission (excitation = 295 nm; emission = 34 〇 nm, 16 nm bandpass) were used to determine the association rate. "Biological activity" and "biological characteristics" with respect to the polypeptide of the present invention means the ability to bind to a biomolecule unless otherwise stated. "Biomolecule" means a nucleic acid, a protein, a carbohydrate, a lipid, and combinations thereof. In one embodiment, the biomolecule is naturally occurring. "Isolated" when used to describe the various antibodies disclosed herein, means an antibody that has been identified and isolated and/or recovered from the cell or cell culture in which it is expressed. For polypeptides, the contaminating component of the natural environment is a substance that will generally be used for diagnostic or therapeutic purposes, and may include enzymes, hormones, and other proteinaceous or non-protein f solutes. In a preferred embodiment, the anti-boat will be purified to (1) to a degree sufficient to obtain an N-terminal or internal amino acid sequence of 15 residues by using a rotary cup sequencer; or (2) using a library Mussing is preferably obtained by SDS_PAGE under non-reducing or reducing conditions. Since at least one component of the polypeptide's natural environment, the antibody thus isolated includes an antibody in situ within the recombinant cell. 142552.doc -40· 201011047 However, typically the isolated polypeptide will be prepared by at least one purification step. The term "control sequence" refers to a DNA sequence necessary for the expression of a coding sequence operably linked within a particular host organism. Suitable for prokaryotic organisms, the 3⁄4 sequence includes, for example, a promoter, a manipulation sequence as appropriate, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. When a nucleic acid is functionally related to another nucleic acid sequence, the nucleic acid is "operably linked" as an example, and the DNA of the presequence or secretory leader sequence is expressed as a precursor protein involved in the secretion of the polypeptide. Is operably linked to the polypeptide 2 DNA; the promoter or enhancer is operably linked to the coding sequence if it affects the transcription of the coding sequence; or the ribosome binding site is mapped to facilitate translation It is operatively connected to the coding sequence. Generally, "operably linked" means that the DNA sequences being ligated are contiguous and, in the case of a secretory leader sequence, contiguous and in the reading phase. However, enhancers do not have to be contiguous. The ligation is achieved by conjugation to a suitable restriction site. If the isotopes are not present, synthetic oligonucleotide linkers or linkers are used according to conventional practice. The "amino acid sequence identity number (/〇)" for the polypeptide sequence identified herein is defined as the alignment sequence and the introduction of a gap (if necessary) to achieve the maximum sequence identity percentage, and not When any conservative substitution is considered to be part of sequence identity, the percentage of amino acid residues in the same candidate sequence as the amino acid residue in the sequence being compared. Alignment for the purpose of determining percent identity of amino acid sequences can be accomplished in a variety of ways well known in the art, for example, using publicly available computer software such as 142552.doc -41 - 201011047 BLAST, BLAST-2, ALIGN Or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for the measurement alignment, including any algorithms needed to achieve maximum alignment over the full length sequence being compared. However, for the purposes of this document, the amino acid sequence identity % value is generated using a sequence comparing the computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc. and the source code has been filed with the user's file at the US Copyright Office (US Copyright Office, Washington DC, 20559), which is registered under US Copyright Registration No. TXU5 10087. . ALIGN-2 program by Genentech, Inc. (South San

Francisco, California) is publicly available. The ALIGN-2 program should be compiled for UNIX operating systems, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are unchanged. Unless otherwise indicated, the amino acid sequences described herein are adjacent amino acid sequences. According to the present invention, a "structurally dissimilar" biomolecule refers to a biomolecule (protein, nucleic acid, lipid, carbohydrate, etc.) that does not belong to the same class, or, for example, when referring to a protein, has less than 60% compared to each other. Amino acid identity, less than 50% amino acid identity, less than 4% amino acid identity, less than 30% amino acid identity, less than 20% amino acid identity or less than 10 % amino acid identity. Those skilled in the art can readily determine the "stringency" of the hybridization reaction and are typically empirically calculated based on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper bonding, while shorter probes require lower temperatures. Hybridization is generally determined by the ability of the denatured DNA to re-adhere when the complementary strand is present in an environment below its melting temperature of 142552.doc -42 - 201011047. The higher the degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Therefore, it is concluded that the relatively souther temperature will tend to make the reaction conditions more stringent, and therefore the lower relative temperature tends to make the reaction conditions less stringent. For additional details and explanations on the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular.

Biology, Wiley Interscience Publishers, (1995).

As defined herein, "stringent conditions" or "high stringency conditions" can be identified by the following requirements: (1) low ionic strength and high temperature for washing, such as 0.015 bismuth sodium sulphate / 0.0015 Μ sodium citrate / 0.1% Sodium lauryl sulfate at 50 ° C; (2) a denaturing agent (such as formamide) during hybridization, for example with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone 50% (v/v) guanamine/50 mM sodium phosphate buffer (pH 6.5) with 750 mM sodium sulphate, 75 mM sodium citrate at 42 ° C; or (3) at 42 ° C Next, using 50% metformin, 5xSSC (0.75 M NaCh 0.075 Μ sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 DDenhardt's solution, ultrasonic wave The solution was treated overnight in a solution of fish sperm DNA (50 pg/ml), 0.1% SDS and 10% dextran sulfate, and washed at 42 ° C in 〇.2xSSC (sodium gasification / sodium citrate) 10 Minutes were then washed for 10 minutes at 55 ° C in a high stringency wash consisting of O.lxSSC containing EDTA. "Moderately stringent conditions" can be found in Sambrook et al., Molecular

Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, for identification, and includes the use of washing solutions and hybridization conditions (eg, temperature) that are less stringent than those described above in 142552.doc -43-201011047. , ionic strength and %SDS). An example of moderately stringent conditions is overnight incubation at 37 ° C in a solution containing the following: 20% guanamine, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6) , 5x Down's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA; then the filter was washed in lxSSC at about 37 to 50 °C. Those skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length and the like. Antibody "effector function" refers to the biological activity attributable to the Fc region of the antibody (the native sequence Fc region or the amino acid sequence variant Fc region) and which varies with the antibody isotype. Examples of antibody effector functions include: Clq binding and complementation-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors) And B cell activation. "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form that is present on specific cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages). The secreted Ig bound by the Fc receptor (FcR) enables the cytotoxic effector cells to specifically bind to the target cell bearing the antigen and subsequently kill the target cell with the cytotoxin. The antibody is "equipped" with cytotoxic cells and is completely required for killing. Primary cells (NK cells) used to mediate ADCC exhibit only FcyRIII, whereas monocytes exhibit FcyRI, FcyRII, and FcyRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). For the evaluation of the ADCC activity of the 142552.doc • 44· 201011047 molecule, an in vitro ADCC assay can be performed, such as the assay described in U.S. Patent No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model as disclosed in Clynes et al. (Proc. Natl. Acad. Sci. USA) 95:652-656 (1998). "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferably, the FcR is a native sequence human FcR. Furthermore, preferred FcRs are FcRs (y receptors) that bind to IgG antibodies and include receptors for the FcyRI, FcyRII, and FcyRIII subclasses, including dual gene variants of these receptors, as well as alternative splicing forms. FcyRII receptors include FcyRIIA ("Activated Receptor") and • FcYRIIB ("Inhibitory Receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domain. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibitory element (ITIM) in its cytoplasmic domain (see M. Review in Damron, Annu. Rev. Immunol. 15:203-234 (1997)). The FcR line is reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin· Med 126:330-41 (1995). The term "FcR" as used herein encompasses other FcRs that are to be identified in the future. The term also encompasses the nascent receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)). 142552.doc -45- 201011047 "Human effector cells" are white blood cells that exhibit one or more FcRs and perform effector functions. Preferably, the cells exhibit at least FcyRIII and perform an ADCC effect function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. Effector cells can be isolated from natural sources such as blood. "Complement-dependent cytotoxicity" or "CDC" is a measure of the dissolution of target cells in the presence of complement. Activation of a typical complement pathway is initiated by binding the first component of the complement system (Clq) to an antibody that binds to its cognate antigen (subject to a suitable subclass). To evaluate the activation of the sputum, you can perform (for example)

CDC assay as described in Gazzano-Santoro et al, J. Immunol. Methods, 202: 163 (1996). The term "therapeutically effective amount" refers to an amount of an antibody or antibody fragment that treats a disease or condition in an individual. In the case of a tumor (eg, a cancerous tumor), a therapeutically effective amount of an antibody or antibody fragment (eg, a multiplex-specific antibody or antibody fragment that specifically binds to HER2 and VEGF) can reduce the number of cancer cells; reduce the size of the primary tumor Inhibiting (ie, to some extent slowing down and preferably terminating) cancer cells infiltrating into peripheral organs; inhibiting (ie, slowing down and better terminating) tumor metastasis; inhibiting tumor growth to some extent And/or to some extent alleviate one or more symptoms associated with the condition.

(TTP), response rate (RR), sex and/or cytotoxicity. About cancer treatment Survival duration, time to disease progression Duration of response and/or quality of life 142552.doc 201011047 Test in vivo efficacy. "Reducing or suppressing" means reducing the overall reduction of the overall reduction of 20% or more, more preferably 5% or more, and optimally 75%, 85%, 9G%, 95% or more: > 'Or inhibition may refer to the presence or size of a cancer metastasis, the size of a primary tumor, or the size or number of blood vessels in an angiogenic condition. The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is generally characterized by unregulated cell growth/proliferation. Examples of such definitions include benign and malignant cancers including, but not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung glands. 'Cancer, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer , glioblastoma, glioma, cervical cancer, nest cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (eg Renal cells _ cancer), liver cancer, prostate cancer, genital cancer, squamous adenocarcinoma, liver cancer, anal cancer penis cancer, melanoma and various types of head and neck cancer. "early cancer" means a cancer that is non-invasive or metastatic or classified as a sputum, 〗 〖 or π stage cancer. "Operational cancer" refers to a condition or growth that usually precedes cancer or develops into cancer. "Non-metastatic" means that the cancer is benign or remains at the primary site and does not penetrate into the lymphatic or vascular system or in tissues other than the primary site. Non-metastatic cancer is usually any cancer, which is sputum, stage 1 or 11 cancer 142552.doc •47· 201011047 and sometimes stage III cancer. "A non-malignant disease or condition involving abnormal activation of job 2" is a condition which does not include a cancer in which abnormal activation of HER2 occurs in cells or tissues of an individual having or predisposed to a disease or condition. Examples of such diseases or conditions include autoimmune diseases (e.g., psoriasis), see definitions below; endometriosis; scleroderma; restenosis; polyps, such as colon 2 meat, nasal polyps or gastrointestinal polyps; Tumor; respiratory disease (eg chronic bronchitis 'asthma (including acute asthma and allergic asthma), cystic fibrosis, bronchiectasis, allergic or other rhinitis or sinusitis, alpha 1 anti-trypsin deficiency, ", emphysema, Pulmonary fibrosis or tracheal hyperactivity, chronic obstructive pulmonary disease and chronic obstructive pulmonary disease); cholecystitis; = neurofibromatosis; polycystic kidney disease; m disease; skin disorders including psoriasis and dermatitis; vascular disease; Abnormal proliferation of epithelial cells; gastrointestinal cancer; Menetrier's disease, secreted adenoma or protein loss syndrome; nephropathy; also develops disorders; ophthalmic diseases such as age-related macular degeneration, hypothetical ophthalmic tissue bacteriostatic disorders, origin Retinal neovascularization, retinal angiogenesis, diabetic visual network Disease or age-related macular degeneration; bone-related lesions, such as osteoarthritis, rickets and osteoporosis; damage after cerebral ischemic events; fibrosis or edema disease, such as cirrhosis, pulmonary fibrosis, sarcoma, Thyroiditis, systemic hyperviscosity syndrome, Osh Weber-Rendu disease, chronic obstructive pulmonary disease or burns, trauma, radiation, impact, hypoxic or post-ischemic edema; skin allergic reaction; diabetic retinopathy and diabetes Kidney disease; Guillain-Barre syndrome 142552.doc -48- 201011047 (Guillain-Barre syndrome); graft versus host disease or transplant rejection; Paget's disease; bone or joint inflammation; photoaging ( For example, caused by UV radiation on human skin; benign prostatic hypertrophy; characteristic microbial infection 'includes selected from the group consisting of a knee virus, a hantavirus, a Borrelia burgdorferi, and a Yersinia spp. .) and microbial pathogens of B. rdetella pertussis; thrombus caused by platelet aggregation; reproductive conditions Such as endometriosis, ovarian hyperstimulation syndrome, early sputum, dysfunctional uterus withdrawal or menorrhagia, synovitis; atherectomy; acute and chronic nephropathy (including proliferative spheroids) Nephritis and diabetic-induced kidney disease; eczema; hypertrophic scar formation, endotoxin shock and fungal infection; familial adenoma; polyposis; neurodegenerative diseases (eg Alzheimer's disease (Alzheimer, s • disease) ), AIDS-related dementia, Parkinson's disease (sports atrophic lateral sclerosis, retinitis pigmentosa, vertebral muscle atrophy, and cerebellar degeneration); myelodysplastic syndrome; regenerative disorder • anemia Ischemic injury; _, renal or liver fibrosis; cell-mediated allergic disease; infant hypertrophic pyloric stenosis; urinary tract obstructive syndrome; psoriasis 'arthritis and Hashimoto's thyroiditis (Hasim〇t〇 , s thyroiditis). As used herein, an 'autoimmune disease' is a disease or condition produced by an individual's own tissue and directed against the individual's own tissue, or a co-segregation thereof, or a symptom or resulting condition. Examples of autoimmune diseases or conditions include, but are not limited to, arthritis (rheumatoid arthritis, reading such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, 142552.doc -49- 201011047 Gouty arthritis, chronic inflammatory joints, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis: skin spasm, arthritis, vertebral arthritis and juvenile rheumatoid Arthritis, inflammation, chronic progressive arthritis, malformation arthritis, chronic primary polyarthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin disease, psoriasis (such as plaque Psoriasis, psoriasis, pustular psoriasis and psoriasis of nails), dermatitis (including contact dermatitis, chronic contact dermatitis, atopic dermatitis, allergic contact dermatitis, herpes-like dermatitis and atopic dermatitis), X-linked high IgM syndrome lkmed hyper igM syndr〇me), urticaria (such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria) Polymyositis/dermatomyositis, adolescent dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis (such as systemic sclerosis, multiple sclerosis (MS), Such as optic neuromyelitis MS (sino-optic MS), primary progressive ms (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis And ataxia sclerosis), inflammatory bowel disease (IBD) (eg 'cr〇hn's disease, autoimmune-mediated gastrointestinal disease, colitis (such as ulcerative colitis) (ulcerative colitis), ulcerative colitis (e〇litis ulcerosa), microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis and transmural colitis) and autoimmune inflammatory bowel Pathology), gangrenous pyoderma, nodular erythema, primary sclerosing cholangitis, upper scleritis, respiratory distress syndrome (including adult or acute respiratory distress syndrome (ARDS)), 142552.doc -50- 201011047 Meningitis, grapes Inflammation of all or part of the membrane, iritis, choroiditis, autoimmune blood disease, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases (such as severe allergies and allergic and atopic rhinitis) , encephalitis (such as Rasmussen, s encephaliUs) and marginal encephalitis and / or brainstem encephalitis, uveitis (such as anterior uveitis, acute anterior uveitis, granulomatous grapes) Membrane inflammation, non-granulomatous uveitis, lens antigenic uveitis, posterior uveitis or autoimmune grapes ▲ eutrophic inflammation, glomerular nephritis (GN) with and without renal disease (such as _ Chronic or acute glomerulonephritis, such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrane proliferative GN or Membranous proliferative GN (MPGN) (including type I and 'sputum type) and rapid progressive GN), allergic conditions, allergic reactions, eczema (including allergic or atopic eczema), asthma (such as Bronchial asthma (asthma bronchiale), bronchial asthma (br〇nchiai Asthma and autoimmune asthma), conditions involving T cell infiltration and chronic inflammatory reactions, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion defects, systemic lupus erythematosus (SLE) or whole body Lupus erythematosus (such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritis, encephalitis, children, non-kidney, kidney) External, discoid, alopecia), adolescent (type 1) diabetes (including childhood insulin-dependent diabetes mellitus (IDDM), adult-type diabetes (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, and Cytokine and T lymphocyte-mediated acute and delayed hypersensitivity-related immune responses, tuberculosis, sarcoidosis, granulosa (including lymphoma-like 142552.doc 51 201011047 granulomatosis 'Wegener's granulomatosis )), granular ball deficiency, vasculitis (including vasculitis (including major vascular vasculitis (including rheumatic polymyalgia and giant cells (Takayasu's )) arteritis), moderate vascular vasculitis (including Kawasaki's disease and nodular polyarteritis), microscopic polyarteritis, CNS vasculitis, necrotizing vasculitis, cutaneous vasculitis Or hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis (such as Churg-Strauss vasculitis or syndrome (CSS)), temporal arteritis, regeneration Obstructive anemia, autoimmune aplastic anemia, Coombs positive ©❹ anemia, Diamond Blackfan anemia, hemolytic anemia, or immune hemolytic anemia (including autologous Immunohemolytic anemia (AIHA), pernicious anemia, Addison's disease, pure red blood cell anemia or hypoplasia (PRCA), Factor VIII deficiency, type A jk, autoimmune Neutrophce reduction, whole blood cell reduction, leukopenia, diseases involving leukocyte oozing, CNS inflammatory conditions, multiple organ injury syndrome (such as multiple organ damage secondary to sepsis, trauma or bleeding) Syndrome), antigen-antibody recombination-mediated disease, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bech's or Behcet's disease, Castleman's syndrome ), Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, and so on (such as Bullous pemphigoid and pemphigoid), pemphigus (including common days 142552.doc -52- 201011047 acne, deciduous pemphigus, pemphigus pemphigoid and erythematous pemphigus), autologous Immune polyendocrine disease, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, optic neuromyelitis, polyneuropathy, chronic neuropathy (such as IgM polyneuropathy or igM-mediated Neuropathy), thrombocytopenia (eg 'progressively from patients with myocardial infarction) (including embolic) Small plate reduced purpura (TTP) and autoimmune or immune-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura (ITp) (including chronic or acute sputum), autoimmune testis and ovary Diseases (including autoimmune testicular and oophoritis), primary hypothyroidism, parathyroid hypoxia, autoimmune endocrine diseases (including thyroiditis, such as autoimmune thyroiditis, Hashimoto's disease, Chronic thyroiditis (Hashimoto, s thyr〇iditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglygonism Symptoms (such as autoimmune polyadenotrophic syndrome (or multi-adenotrophic endocrine disorders)), paraneoplastic syndromes (including neuro-paraneoplastic syndromes, such as Lambert-Eaton myasthenic syndr〇) Or Eton n-Lambert syndr〇me), zombie syndrome (10) ffman or sUff-person syndr〇me), Myelitis (such as allergic cerebrospinal inflammatory disease and experimental allergic rhinitis _), myasthenia gravis (such as thymoma-associated myasthenia gravis), cerebellar degeneration, neuromuscular rigidity, visual ocular dysfunction or Vision eye lining muscle array climbing syndrome (_) and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome (SheeWs scan. (4), autologous 142552.doc -53- 201011047 immune hepatitis, chronic hepatitis, lupus-like hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia, obstructive bronchiolitis (non-transplantation) with NSIP, Gu Li'an - white end syndrome

Group (GuUlain-Barr0 syndrome), Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA skin disease, primary biliary cirrhosis, pulmonary cirrhosis, autoimmune bowel Symptoms, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory stomatitis diarrhea, idiopathic steatorrhea, cryoglobulinemia, muscle atrophic lateral cord Hardening (ALS; L〇u Gehrig's disease), coronary artery disease, autoimmune ear disease (such as autoimmune inner ear disease (AIED)), autoimmune hearing loss, visual ocular array Ankle syndrome (OMS), polychondritis (such as refractory or recurrent polychondritis), alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis ( Including single-cell B-cell lymphadenopathy (such as benign individual gamma globulin disease and unexplained single globulin globulin (MGUS)), peripheral neuropathy, paraneoplastic syndrome, ion channel disease (such as epilepsy, partial headache, Arrhythmia, muscle disease, deafness, periodic anesthesia and CNS ion channel disease), autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine eye disease, uveal retina Inflammation, choroidal retinitis, autoimmune liver disease, muscle fiber pain, multiple-time endocrine failure, Schmidt group (sehmi_ café), adrenal gland inflammation, gastric atrophy, Alzheimer's disease, de- _ disease (such as autoimmune Demyelinating disease, diabetic nephropathy, Dresler's syndrome (Dressler, s syndr〇me), alopecia areata, crest syndrome (5) Shen 142552.doc -54- 201011047

Disease, Raynaud's phenomenon, abnormal esophageal function, fingertip scleroderma and telangiectasia, autoimmune infertility for men and women, mixed connective tissue disease, Chagas* disease, Rheumatic fever, habitual abortion, peasant lung, erythema multiforme, post-cardiac surgery syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous vasculitis, benign lymph Cellular vasculitis, Alport's syndrome, alveolitis (such as allergic alveolitis and fibrotic alveolitis), interstitial lung disease, transfusion reaction, leprosy, cancer, leishmaniasis (leishmaniasis), trypanosomiasis (kypanosomiasis), schistosomiasis, ascariasis, sputum disease, Samter's syndrome, Caplan's syndrome, dengue, endocardium Inflammation, internal myocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, persistent bulging red , fetal red blood cell blastosis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, ciliary body inflammation (such as chronic ciliary inflammatory disease) , heterochromic ciliary body inflammation, iridocyclitis or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) Infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection (Epstein-Barr virus infection), epidemic legitis, Evan's syndrome, autoimmune 142552.doc -55- 201011047 Glandular exhaustion, Sydenham's chorea, chain Nephritis, thromboangiitis, scorpion venom, spinal cord hernia, chorioitis, giant cell polymyalgia, endocrine eye disease, chronic allergic pneumonia, dry cornea after cochlear infection Conjunctivitis, epidemic keratoconjunctivitis, idiopathic renal syndrome, minimally variable nephropathy, benign familial and ischemic/reperfusion injury, autoimmune of the retina, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, Aphthous, aphthous stomatitis, arteriosclerotic disease, aspermiogenese, autoimmune hemolysis, Boeck's disease, condensed globulinemia, Dupuytren's contracture, Lens allergic endophthalmitis, irritable bowel, jatropha nodular erythema, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sense Phonological hearing loss, paroxysmal hemoglobinuria, gonadal dystrophy, colitis, leukopenia, infectious mononuclear hematocytosis, transverse myelitis, primary idiopathic mucinous edema, kidney disease, father Ophthalmia symphatica, granulomatous smear, pancreatitis, acute polyradiculitis, gangrenous pyoderma, Quervain's thyreoiditis, acquired spleen atrophy, infertility due to anti-sperm antibodies, non-malignant thymoma, leukoplakia, SCID and disease associated with Eberstein-Barr virus (EpStein-Barr virus- Associated disease), acquired immunodeficiency syndrome (AIDS), parasitic diseases (such as Leishmania), toxic shock syndrome, food poisoning, conditions involving T cell infiltration, white blood cell adhesion defects, and cytokines and T_lymphocyte-mediated acute and delayed 142552.doc •56- 201011047 Allergic-related immune system response, diseases involving leukocyte exudation, multiple @官-injury syndrome, antigen-antibody complex-mediated disease, anti-kidney Basement membrane disease, allergic neuritis 'autoimmune endocrine disease, nestitis, primary mucinous edema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, Kidney syndrome, Tengdao inflammation, multiple endocrine failure, peripheral neuropathy, autoimmune polyadenosine syndrome, adult type Sexual hypothyroidism (A〇IH), total alopecia, dilated cardiomyopathy, acquired bullous epidermis (epidermuolisi's bullosa aequisita, EBA), hemochromatosis, myocarditis, renal disease, primary sclerosis Cholangitis, purulent or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, anterior lobe, humerus or sphenoid sinusitis, eosinophilia • blood cell-related disorders (such as eosinophilia, pulmonary invasive eosinophilia Blood • Ball increase, eosinophilic myalgia syndrome, Loffler's syndr〇me, chronic eosinophilic pneumonia, tropical lung eosinophilia, bronchial pneumonia, aspergillosis Or granuloma with eosinophils), severe allergies, seronegative spinal arthritis, multiendocrine autoimmune disease, sclerosing cholangitis, sclera, upper sclera, chronic mucocutaneous candidiasis, Bruton's syndrome (Brut 〇n, s syndrome); infants with transient hypogammaglobulinemia; Wiskott-Aldrich syndrome, ataxia telangiectasia, Autoimmune disorders associated with prion protein disease, rheumatism, neuropathy, ischemia-reperfusion disorders, reduced stress response, vascular dysfunction, vasodilation, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia and Diseases associated with angiogenesis, allergies, spheroid nephritis, reperfusion injury, heart 142552.doc -57- 201011047 Reperfusion injury of muscle or other tissues, skin disease with acute inflammatory components, acute suppurative cerebral ridge Membrane inflammation or other inflammatory diseases of the mid-frame nervous system, inflammatory diseases of the eyes and eyelids, syndromes associated with granule ball infusion, cytokine-induced toxicity, acute severe inflammation, chronic refractory inflammation, pyelonephritis, cirrhosis, diabetic retinopathy , diabetic aortic disease, intra-arterial hyperplasia, peptic ulcer, heart disease and endometriosis. "Anti-angiogenic agent" or "angiogenesis inhibitor" refers to small molecular weight substances, polynucleotides, peptides, isolated proteins, recombinants that directly or indirectly inhibit angiogenesis, angiogenesis or unwanted vascular permeability. Protein, antibody or binding protein or fusion protein thereof. For example, the anti-angiogenic agent is an antibody or an anti-angiogenic agent other than an antagonist, such as an anti-VEGF antibody (eg, bevacizumab (AVASTIN7), bHl, bHl-44, bHl) -81), an antibody against the VEGF receptor, a small molecule that blocks VEGF receptor signaling (eg, PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinib malate), AMG706). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin and the like. See, for example, Klagsbrun and D'Amore, /! «««· J^v. Corpse 53:217-39 (1991) 'Streit and Detmar, 22:3172-3179 (2003) (for example, Table 3 lists malignant melanoma Anti-angiogenic therapy); Ferrara and Alital0,

Mdic/Plus 5(12): 1359-1364 (1999); Tonini et al., (3) Iga, 22:6549-6556 (2003) (for example, Table 2 lists anti-angiogenic factors), and Sato site X (10) 8:200·206 (2〇〇3) (for example, Table 1 lists the antibiotics in the clinical trials). The sputum tube & dysregulation can cause a condition that can be treated by the compositions and methods of the invention. Such conditions include both non-neoplastic and neoplastic conditions.

The term "cytotoxic agent" as used herein refers to a substance which inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioisotopes (eg, radioactive isotopes of At211, I131, 1丨25, Y90, Re186, Re188, Sm153, Bi212, Ra223, P32, and Lu); chemotherapeutic agents, such as methotrexate, Adriamicin, vinca alkaloid (vinchun, new vincristine, vinblastine, etoposide), doxorubicin, melphalan ), mitomycin C, chlorambucil, daunorubicin or other intervening agents, enzymes and fragments thereof (such as nucleases), antibiotics and toxins (such as bacteria) Small molecule toxins or enzymatically active toxins, including fungal, plant or animal derived sources, including fragments and/or variants thereof, as well as various antitumor or anticancer agents disclosed herein. Other cytotoxic agents are described herein. Tumor killing agents cause destruction of tumor cells. 0 "Chemotherapeutic agents" are compounds suitable for the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; succinic acid vinegars such as busulfan, improsulfan and piperazine Shufan (pip0suifan); aziridines, such as benzodopa, carb〇quone, meturedopa and uredopa; exoethylenimines and Methyl melamines, including altretamine, tri-ethyl melamine 142552.doc -59- 201011047 (triethylenemelamine), trietylenephosphoramide, tri-ethyl thio Triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); δ-9- Deltaa-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapaehol; colchicine; betulinic acid (betulinic acid); camptothecin (including synthetic analog topography) (topotecan) (HYCAMTIN®), CPT-11 (irinotecan; CAMPTOSAR®, B. sylvestre, scopolectin, and 9-aminopyridine); bryostatin ); calistatin (CCallystatin); CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogues); Podophyllotoxin); podophyllinic acid; teniposide; cryptophycin (especially Nostoccal cyclic peptide 1 and Nostoccal ring 8); dolphin (dolastatin); Doka Duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin · spongistatin; Nitrogen mustards, such as benzobutyric acid mustard, chlornaphazine, cyclosporin, estramustine, ifosfamide, mechlorethamine, and nitrogen mustard , melphabine, new engembichin, cholesterol to phenylacetate mustard (pheneste Rine), 松龙 142552.doc -60- 201011047 phenylfenac (prednimustine), trofosfamide, urine, uracil mustard; nitrogland, such as arginine mustard ( Carmustine), chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne Antibiotics (for example, calicheamicin, especially calicheamicin gamma 1 (see, eg, Agnew, Chem Inti. Ed Engl, 33: 183-186 (1994)); dynemicin, Including danamycin A; esperamicin; and neocarzinostatin chromophore and related chromophore diacetylene antibiotic chromophore, aclacinomysins, Actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carbofurin

(carabicin), carminomycin, carzinophilin, chromomycinis, dactinomycin, donovan, detorubicin, 6-weight Nitro-5-oxo-L-positral leucine, ADRIAMYCIN® doxorubicin (including morpholinium doxorubicin, cyanomorpholine doxorubicin, 2-pyrrole doxorubicin, and deoxygenated Phytomycin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomyeins (such as mitomycin C) ), mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, Triamcinolone 142552.doc -61 - 201011047 (quelamycin), rodorubicin, streptonigrin, streptozocin, tubercidin, urinary benzene Ubimimex, zinostatin, zorubicin; antimetabolites such as methotrexate 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; bite analogs, such as cyclosporine (ancitabine) 'azacitidine, 6-1-azauridine, carmofur, cytarabine, dideoxyuridine, deoxygenation Doxifluridine, enocitabine, floxuridine; androgens, such as calpressone, dromostanolone propionate, epitisolol ), mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as Folinic acid; vinegar vinegar (aceglatone); acid dish glycosides (aldophosphamide glycoside); aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; Edatraxate; defofamine; colchicine 142552.doc -62- 201011047

(demecolcine); diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate ;; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; (mitoguazone); mitoxantrone; mopidanmol; nitraerine; pentostatin; phennamet; °tb Pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® multi-audio complex (JHS Natural Products, Eugene, OR); propylimine (razoxane); rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2·, 2" - trichlorotriethylamine; trichothecenes (especially Τ-2 toxin, veraculinin A (verracurin) A), roridin A and anguidine; urethan; vindesine (ELDISINE®, FILDESIN®); azene (dacarbazine); Mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara -C"); thiotepa; taxoids such as TAXOL® Pacific Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ); 142552.doc -63- 201011047 ABRAXANETM (excluding Klemophor ( Cremophor)) (ie, paclitaxel engineered nanoparticle formulations of Pacific Paclitaxel) (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® doxetaxel (Rh6ne-Poulenc Rorer, Antony, France); Chloranbucil; gemcitabine (GEMZAR®); 6-stone guanine (6-thioguanine); thioglycan; methotrexate; initial analogue, such as cisplatin ) and card turned (carboplatin); Changchun flower test (vinblast Ine) (VELBAN®); beginning; relying on bitter (VP-16); heterocyclic decylamine; mitoxantrone; © Xinchunxin (ONCOVIN®); oxaliplatin; Leucovovin; vinorelbine (NAVELBINE®); Novantrone; edatrexate; daunomycin; Ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); a pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of the two or more of the above, such as CHOP (cyclophosphamide, doxorubicin, periwinkle) Abbreviation for the combination therapy of neobase with prednisolone) and FOLFOX (abbreviation for the treatment regimen of oxaliplatin (ELOXATINTM) combined with 5-FU and formic acid). Also included in this definition are anti-hormonal agents which are used to modulate, reduce, block or inhibit the action of hormones which promote cancer growth and which are often in the form of systemic or holistic treatment. It can be a hormone itself. Examples include anti-estrogen and selection 142552.doc 201011047 sex estrogen receptor modulator (SERM), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene ( Raloxifene), droloxifene, 4- tacroxoxene, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® Torre Toremifene; anti-progesterone; estrogen by a few adjustments (ERD); agents that inhibit or stop the action of the ovaries, such as lutein releasing hormone (LHRH) agonists, such as LUPRON® and ELIGARD® acetate Leuprolide acetate, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide, nitrite Nilutamide and bicalutamide; and aromatase inhibitors that inhibit the regulation of estrogen production by the estrogen in the adrenal gland, such as 4(5)-miso, aminogglutamine, MEGASE® quercetin acetate (megestrol a Cetate), AROMASIN® exemestane, fulmestan

(formestanie), fadrozole, RIVISOR® vorozole, FEMARA® (letrozole) and ARIMIDEX® anastrozole. In addition, this definition of chemotherapeutic agent includes: bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® Zoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate or ACTONEL® risedronate Salt (risedronate); and troxacitabine 142552.doc -65- 201011047 (troxacitabine) (l,3-dioxolan nucleoside sulphonic biting analog); antisense oligonucleotides, especially inhibitory genes involved Antisense nucleotides expressed in signaling pathways in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccines and genes Therapeutic vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (ErbB-2 and EGFR) Tyrosine kinase small molecule inhibitor, also known as GW572016); Any above one of the acceptable pharmaceutically acceptable salts, acids or derivatives thereof. "Growth inhibitor" as used herein refers to a compound or composition that inhibits cell growth in vitro or in vivo. Therefore, the growth inhibitor can be a person who significantly reduces the percentage of cells in the S phase. Examples of growth inhibitors include agents that block cell cycle progression (outside the S phase), such as agents that induce G1 arrest and stagnation. Typical sputum blockers include vinca (eg, Changchun and Changchun), taxane, and topoisomerase type II inhibitors, such as doxorubicin, epirubicin, and Donovan , etoposide and bleomycin. Suppressing agents that cause G1 stagnation also cause S-phase arrest, such as DNA burning agents such as tamoxifen, prednisone, imipenem, nitrogen mustard, cisplatin, amidoxime, 5-fluorourine Pyridine and ara-C. Additional information can be found in The Molecular Basis of Cancer, edited by Mendelsohn and Israel, Chapter 1, Murakami et al., entitled [Cell cycle regulation, oncogenes, and antineoplastic drugs] (WB Saunders: Philadelphia, 1995), especially on page 13. . Taxanes (pacific paclitaxel 142552.doc 201011047 and docetaxel) are anticancer drugs derived from yew. TAXOTERE® (Rhone-Poulenc Rorer) from European yew is a semi-synthetic analog of TAXOL® (Bristol-Myers Squibb). Pacific paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in inhibition of mitosis in cells.

As used herein, "anti-cancer therapy" refers to the treatment of reducing or inhibiting cancer in an individual. Examples of anti-cancer therapies include cytotoxic radiation therapy and administration to a subject a therapeutically effective amount of a cytotoxic agent, a chemotherapeutic agent, a growth inhibitor, a cancer vaccine, an angiogenesis inhibitor, a prodrug, a cytokine, a cytokine antagonist, a cortex Steroids, immunosuppressants, antiemetics, antibodies or antibody fragments or analgesics. The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance which is less cytotoxic to tumor cells than the parent drug and which can be enzymatically activated or converted to A parent form that is more active. See, for example, Wilman, [Prodrugs in Cancer Chemotherapy] Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al, [Prodrugs: A Chemical Approach to Targeted Drug Delivery] Directed Drug Delivery, Borchardt Et al. (eds.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, prodrug-containing prodrugs, thiophosphate-containing prodrugs, prosulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylation prodrugs Pre-drug containing β·indoleamine, containing optionally substituted phenoxyethyl 142552.doc -67- 201011047 proguanamine prodrug or pre-drug containing phenethylamine as appropriate, can be converted into activity Strong cytotoxic free drug 5-fluorocytosine and other 5-fluorouridine prodrugs. Examples of cytotoxic drugs which may be derivatized into a prodrug form suitable for use in the present invention include, but are not limited to, the chemotherapeutic agents described above. The term "cytokine" is a generic term for a protein released by a cell population that acts as an intracellular mediator on another cell. Examples of such cytokines are lymphokines, mononuclear hormones and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone (HGH), N-methionine-based human growth hormone and bovine growth hormone; parathyroid hormone; scorpion stimulating hormone; ginseng phosphatase; proinsulin; relaxin; Relaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH) 'epidermal growth factor (EGF); liver growth factor; fibroblast growth. FGF) ·, prolactin; placental lactogen; tumor necrosis factor "and tumor necrosis factor_β; mullerian_inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; Endothelial growth factor; integrin; thrombopoietin (τρ〇); nerve growth factor, such as NGF_a; platelet growth factor; transforming growth factor (TGF) such as TGF_a and TGF p; insulin-like growth factor heart and insulin-like growth factor- II; erythropoietin (Ep〇); osteoinductive factors; interferons such as interferon-a, interferon and interferon_γ; community stimulating factor (CSF) 'such as macrophage_CSF ( M_CSF); granulocyte-macrophage·CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukin (IL), such as 1L_1, IL_la, IL-2, IL-3, IL- 4. IL-5, IL-6, IL-7, IL-8, IL-9, iL-i〇, IL_U, IL_12; tumor necrosis factor, such as 142552.doc -68- 201011047 TNF-α or TNF-β And other polypeptide factors including LIF and kit ligands (KL). The term cytokine as used herein includes a biologically active equivalent of a protein derived from a natural source or from a recombinant cell culture and a native sequence cytokine. "Cyto Hormone Antagonist" means a molecule that partially or completely blocks, inhibits or neutralizes the biological activity of at least one cytokine. For example, a cytokine antagonist can inhibit cytokine activity by inhibiting cytokine expression and/or secretion or by binding to a cytokine or cytokine receptor. Cytokine antagonists include antibodies that bind to cytokines or cytokine receptors, synthetic or native sequence peptides, immunoadhesins, and small molecule antagonists. The cytokine antagonist is bound or fused to the cytotoxic agent as appropriate. Exemplary TNF antagonists are etanercept (ENBREL®), infliximab (REMICADE®), and adalimumab (HUMIRAtm).

The term "immunosuppressive agent" as used herein refers to a substance that acts to inhibit or mask the immune system of a treated individual. This may include inhibiting cytokine production, downregulating or inhibiting the expression of the MHC antigen by the autoantigen. Examples of immunosuppressive agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); mycophenolate mofetil, such as CELLCEPT®; sulfur 11 sitting Azathioprine (IMURAN®, AZASAN®/6-mercaptopurine; bromocryptine; danazol; dapsone; glutaraldehyde (which masks MHC antigens, eg U.S. Patent No. 4,120,649; anti-genetic antibodies against MHC antigens and MHC fragments; cyclosporine 142552.doc-69-201011047 A (cyclosporin A); steroids such as corticosteroids and glucocorticosteroids, For example, prednisone, prednisolone such as PEDIAPRED® (prednisolone sodium phosphate) or ORAPRED® (prednisolone sodium phosphate oral solution), methylprednisolone and dexamethasone (dexamethasone) Amidoxime (oral or subcutaneous) (RHEUMATREX®, TREXALLTM); hydroxycloroquine / chloroquine • sulfasalazine; leflunomide (leflunomide); cytokine or cytokine receptor antagonist package Including anti-interferon-γ, anti-interferon-β or anti-interferon-α antibody, anti-tumor necrosis factor-α antibody (infliximab or adalimumab), anti-TNFa immunoadhesin (ENBREL®, ena Sigma, anti-TNF-β antibody, anti-interleukin-2 antibody and anti-IL-2 receptor antibody; anti-LFA-1 antibody, including anti-CD11a and anti-CD18 antibody; anti-L3T4 antibody; heterologous anti-lymph Cytoglobulin; multi-strain or pan-T antibody or monoclonal anti-CD3 or anti-CD4/CD4a antibody; soluble peptide containing LFA-3 binding domain (WO 1990/08187, published July 26, 1990); Streptococcus Kinase; TGF-β; Streptococcus deoxyribonuclease; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Patent No. 5,114,721); T-cell receptor fragment (Offner et al. 8 <^611, 6, 251, 430-432 (1991); WO 1990/11294; Ianeway, Nature, 341: 482 (1989); and WO 1991/01133); T cell receptor antibody (EP 340, 109), such as T10B9; cyclophosphamide (CYTOXAN®); amine benzoquinone; Amine (CUPRIMINE®); switched plasma cells; or immune 142552.doc -70- 201011047 intravenous immunoglobulin (IVIG). The immunosuppressive agents may be used alone or in combination with each other, especially in combination with a steroid and another immunosuppressive agent or in combination with a maintenance dose of a non-steroidal agent to reduce the need for steroids. "Analgesic" means A drug that inhibits or suppresses pain in an individual. Exemplary analgesics include non-steroidal anti-inflammatory drugs (NSAIDs), including ibuprofen (MOTRIN®), naproxen (NAPROSYN®), acetylated salicylic acid, indomethacin, and sedatives. Sulmedac and tolmetin include salts and derivatives, as well as other drugs used to reduce possible puncture pain including anticonvulsants (gabapentin, phenyloin) ), carbamazepine or tricyclic antidepressants. Specific examples include acetaminophen, aspirin, amitriptyline (amitriptyline, ELAVIL®), carbamazepine (TEGRETOL®), benzene

Phenyltoin (DILANTIN®), gabapentin (NEURONTIN®), (E)-N-vanillyl-8-mercapto-6-neuramide (CAPSAICIN®) or a nerve blocker. "Corticosteroid" refers to any of a number of synthetic or naturally occurring substances having the general chemical structure of a steroid that mimics or augments the action of a naturally occurring corticosteroid. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, triamcinolone, and betamethasone. A "cancer vaccine" as used herein is a composition that stimulates an immune response against cancer in an individual. Cancer vaccines are generally composed of cancer-related substances or cells (antigens) that can be homologous (from themselves) or allogeneic (from others) and other components (such as adjuvants) to further Stimulates and enhances the immune response against the antigen. Cancer vaccines desirably result in stimulating an immune system of an individual to produce antibodies against one or several specific antigens' and/or to produce killer tau cells to attack cancer cells having their antigens. As used herein, "cytotoxic radiation therapy" refers to radiation therapy that inhibits or prevents cellular function and/or causes cell destruction. Radiation therapy can include, for example, external beam irradiation or therapy with radiolabeled agents such as antibodies. The term is intended to include the use of radioisotopes (e.g., radioactive isotopes of 'At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, Ra223, P32, and Lu). An "antiemetic" is a compound that reduces or prevents nausea in an individual. Antiemetic compounds include, for example, neurokinin-1 receptor antagonists, 5HT3 receptor antagonists (such as ondansetron, granisetron, tropisetron, and zafi Zhaisetron), GABAB receptor agonist (such as baclofen), corticosteroids (such as dexamethasone, KENALOG®, ARISTOCORT® or NASALIDE®), anti-dopaminergic drugs, Pyrazines (eg, prochlorperazine, fluphenazine, thioridazine, and mesoridazine), dronabinol, metroclopramide ), domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine, and levomepromazine The "individual" is a vertebrate, preferably a mammal, and more preferably a human. 142552.doc -72- 201011047 Mammals include, but are not limited to, farm animals (such as cows), sports animals, pets (such as juveniles, dogs and horses), primates, mice, and rats. Commercially available reagents mentioned in the examples are used according to the manufacturer's instructions unless otherwise stated. The sources of these cells identified by the ATCC registration number in the examples below and throughout the specification are the American Type Culture Collection, Manassas, VA. Unless otherwise indicated, the present invention uses standard procedures for recombinant DNA techniques, such as those described above and in the following textbooks: Sambrook et al., supra; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY" 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: NY, 1990); Harlow et al.

Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al, Current Protocols in Immunology, 1991. Throughout this specification and the scope of the claims, the word "comprising" is to be understood as meaning to include the enumerated integer or integer group, but does not exclude any other integer or integer group. II. Vectors, Host Cells, and Recombinant Methods For the recombinant production of an antibody of the present invention, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further selection (amplification of DNA) or expression. The DNA encoding the antibody is readily isolated and using conventional procedures (e.g., by using an oligonucleotide probe capable of binding to a gene-specific knot 142552.doc-73-201011047 encoding the heavy and light chains of the antibody) sequence. Many vectors are available. The choice of vector will depend, in part, on the host cell to be used. Preferred host cells typically have a source of prokaryote or eukaryote (usually a mammal). It will be appreciated that any homomorphic region, including IgG, igM, IgA, IgD and IgE moieties' can be used for this purpose and that such definitive regions can be obtained from any human or animal species. a. Generation of anti-occupation using prokaryotic host cells i. Vector construction A polynucleotide sequence encoding a polypeptide component of an antibody of the invention can be obtained using standard recombinant techniques. The desired polynucleic acid sequence can be isolated from the antibody-producing cells (such as fusion tumor cells) and sequenced. Alternatively, the polynucleotide can be synthesized using a nucleotide acid synthesizer or PCR technique. After obtaining the sequence encoding the polypeptide, it is inserted into a recombinant vector capable of replicating in a prokaryotic host and exhibiting a heterologous polynucleotide. A wide variety of vectors are available and known in the art for the purposes of the present invention. The choice of a suitable vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed by the vector. Depending on the function of the vector (amplification or expression of the heterologous polynucleotide or both) and the compatibility of the vector with the particular host cell in which it resides, each vector contains various components. Vector components typically include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insertion, and a transcription termination sequence. Generally, plastid vectors containing replicons and control sequences derived from species compatible with the host cell are used in conjunction with such hosts. Vectors typically carry a replication site and a marker sequence that provides phenotypic selection in transformed cells 142552.doc -74· 201011047 column. For example, pBR322 (a plastid derived from an E. coli (five co/z) species) is typically used to transform E. coli. pBR322 contains genes encoding ampicillin 'Amp and tetracycline (Tet) resistance and thus provides an easy way to identify transformed cells. PBR322, its derivatives or other microbial plastids or phages may also contain or be modified to contain promoters which can be used by microorganism organisms to express endogenous proteins. An example of a pBR322 derivative for use in expressing a particular antibody is described in detail in U.S. Patent No. 5,648,237.

In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used in conjunction with such hosts as transformation vectors. For example, a phage such as XGEM.TM_-11 can be used to prepare a recombinant vector that can be used to transform a susceptible host cell such as Escherichia coli LE392. The expression vector of the present invention may comprise two or more promoter-cistronic pairs encoding each polypeptide component. The promoter is an untranslated regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters generally fall into two categories: inducible and constitutive. An inducible promoter is a promoter that initiates an increased amount of cistronic transcription under the control of its response to changes in culture conditions, such as the presence or absence of nutrients or temperature changes. A large number of promoters recognized by a variety of potential host cells are well known. The promoter can be operably linked to the coding light or heavy chain by removing the selected promoter from the source vessel by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Anti-ship. Natural: promoter sequences and multiple, heterologous promoters can be used to guide the expansion and/or performance of the target. - Some implementation of the financial, Xiang (four) promoter, this fairy is as compared with the natural standard 142552.doc -75- 201011047 dry peptide starting, the heterologous promoter usually allows the performance of the target no gene higher transcription and yield high. Promoters suitable for use in prokaryotic hosts include the PhoA promoter, the galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters (such as the tac or trc promoter). However, other promoters that are functional in bacteria, such as other known bacterial or bacteriophage promoters, are also suitable. Nucleotide sequences thereof have been disclosed, thereby enabling those skilled in the art to use ligators or linkers to join cistrons encoding the underlying light and heavy chains (Siebenlist et al., (1980) Cell 20: 269 ) to provide any desired restriction sites. In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptide across the membrane. Typically, the sequence of 彳, 彳5 can be a component of the vector, or it can be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention shall be the ## sequence recognized by the host cell and processed (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced by, for example, a prokaryotic SEQ sequence selected from the group consisting of: phytase, penicillinase , ϊρρ or thermostable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence used in the two cistrons of the expression system is the STII signal sequence or a variant thereof. In another aspect, the production of the immunoglobulin of the present invention can occur in the cytoplasm of the host cell, and thus there is no need for a secretion signal 142552.doc • 76- 201011047 in each cistron. In this regard, both the light and heavy chains of immunoglobulins are expressed, folded, and assembled in the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., E. coli trxB-strain) provide cytoplasmic conditions that facilitate disulfide bond formation, thereby allowing proper folding and assembly of expressed protein subunits (Proba and Pluckthun, Gene, 159:203 (1995)). Prokaryotic host cells suitable for expression of the antibodies of the invention include archaea and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of suitable bacteria include Escherichia (e.g., Escherichia coli), Bacilli (e.g., Bacillus subtilis (Enterobacteria, Enterobacteria, Pseudomonas) species (e.g., P. aeruginosa) P. aeruginosa, Salmonella typhimurium, Serrai/a warcescaws, Klebsiella, Proteus, Shigella (Shigella), Rhizobia, Vitreoscilla or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as the present Host of the invention. Examples of E. coli strains include strain W3 11 〇 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325) and Its derivatives include strain 33D3 (U.S. Patent No. 5,639,635) having the genotype W3110 AfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTA(nmpc-fepE) degP41 kanR. And derivatives thereof (such as Escherichia coli 294 (ATCC 31,446), Escherichia coli B, large 142552.doc -77-201011047 Enterobacteriaceae m76 (ATCC 31,537) and Escherichia coli rv3〇8 (atcc 3U608)) are also Suitable methods for the purpose of illustrating, but not limiting, the derivatives of the above-mentioned bacteria of the genotype (4) are known in the art and are described, for example, in Bass et al., Proteins, 8: 309-3 14 (199 〇). It is generally necessary to consider the replicability of replicons in bacterial cells to select appropriate bacteria. For example, when using well-known plastids (such as PBR322, pBR325, pACYci77 or pKN41〇) When a replicon is provided, E. coli, Serratia, or Salmonella species can be suitably used as a host. Host cells should generally secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can be incorporated into cell cultures as needed. • Antibody Production The host cell is transformed with the above expression vector and cultured in a conventional medium modified as appropriate to induce a promoter, select a transformant or amplify a gene encoding the desired sequence. Transformation means introducing DNA into a prokaryotic host so that the DNA can be replicated as an in vitro element or by a chromosomal component. Depending on the host cell used, 0 is transformed with standard techniques appropriate for such cells. Treatment with gasified calcium is typically used for bacterial cells containing substantial cell wall barriers. Another method of transformation uses polyethylene glycol/DMS. Another technique used is electroporation. Prokaryotic cells used to produce the polypeptides of the invention are grown in a medium known in the art and suitable for culturing selected host cells. Examples of suitable media include Luria broth (LB) plus essential nutrient supplements 142552.doc • 78 · 201011047. In some examples, the medium also contains a selection agent selected based on the construction of the expression vector. 'to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing the ampicillin resistance gene. Any necessary supplements other than carbon, nitrogen and inorganic phosphate sources, either alone or in admixture with another supplement or medium (such as a complex nitrogen source), may also be included. The medium may optionally contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiosulphate. Sugar alcohol. Prokaryotic host cells are cultured at a suitable temperature. For E. coli growth, for example, a preferred temperature is in the range of from about 2 〇〇c to about 39 Torr, more preferably about 25 Å; > c to about 37 C, even more preferably at about 30 eC. The pH of the medium is mainly • Any one of about 5 to about 9 depending on the host organism? 11 values. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0. If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the invention, the ' PhoA promoter is used to control polypeptide transcription. Therefore, the transformed host cells are cultured in a citrate-restricted medium for induction. The sulphate-restricted medium is preferably a C.R.A.P medium (see, for example, Simm〇ns et al, J. Immunol. Methods (2002), 263: 133-147). As is known in the art, a variety of other attracting agents can be used depending on the carrier construct employed. In one embodiment, the expressed polypeptide of the present invention is secreted to the host fine 142552.d〇c -79· 201011047 w~ with and recovered from the cargo. Protein recovery generally involves microbial disruption typically by means such as osmotic shock, ultrasonic treatment or dissolution. Once the cells have shattered, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can be further purified by affinity resin chromatography, and the protein can be transferred to and separated in the medium. The cells can be removed from the nutrient, and the culture supernatant is transiently and concentrated to further: purify the produced protein. The commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay (Western bl〇t assay) can be used to further isolate and identify the expressed polypeptide. In one aspect of the invention, antibody production is carried out in large quantities by fermentation. A variety of large-scale feed batch fermentation procedures can be utilized to generate recombinant proteins. Large-scale fermentation has a capacity of at least 1000 liters, preferably about 1 to 100,000 liters. These fermenters use an impeller agitator to distribute oxygen and nutrients, especially glucose (preferably carbon/energy sources). Small scale fermentation usually refers to fermentation in a fermentor having a volumetric capacity of no more than about 100 liters and a range of from about 1 liter to about 100 liters. In the fermentation method, protein expression is generally initiated after the cells have been grown to the desired density under suitable conditions (e.g., OD550 is about 180-220, at which stage the cells are in the early stationary phase). A variety of inducing agents can be used depending on the carrier construct employed, as is known in the art and as described above. Cells can be grown for a short period of time prior to induction. The cells are usually induced for about 12 to 5 hours' but a longer or shorter induction time can be used. In order to improve the yield and quality of the polypeptide of the present invention, various fermentation conditions can be changed. For example, to improve the proper assembly of the secreted antibody polypeptide and fold 142552.doc • 80 · 201011047, the overexpression accompanying protein can be used ( Additional vectors such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (peptidyl amidoxime cis, trans-isomerase with accompanying protein activity) are used to co-transform host prokaryotic cells. Concomitant proteins have been shown to facilitate proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Bio. Chem. 274: 19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888;

Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol·Microbiol. 39:199- 210. To minimize the proteolysis of the heterologous proteins (especially proteolytically sensitive proteins) exhibited, certain host strains lacking proteolytic enzymes are useful in the present invention. For example, a host cell strain can be modified to achieve a base of a known bacterial protease encoding a protease such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof.

Due to genetic mutations. Some E. coli protease deficient strains are available and described, for example, in J. Ly et al., (1998), Proc. Natl. Acad. Sci. USA 95: 2773-2777; Georgiou et al., U.S. Patent No. 5,264,365 ;Georgiou et al., U.S. Patent No. 5,508,192; Hara et al.

Microbial Drug Resistance, 2: 63-72 (1996). In one embodiment, an E. coli strain that lacks a proteolytic enzyme and exhibits one or more plastid transformations with accompanying proteins is used as a host cell in the expression system of the invention. Iii. Antibody Purification 142552.doc -81 - 201011047 Standard protein purification methods known in the art can be employed. The following procedure is an exemplary suitable purification procedure: fractionation of immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, ceria or cation exchange resin (such as DEAE) chromatography, chromatofocusing, SDS- PAGE, sulfuric acid, and gel filtration using, for example, Sephadex G-75. In one aspect, protein A immobilized on a solid phase is used for immunoaffinity purification of the full length antibody product of the present invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureus which binds to the Fc region of the antibody with high affinity. Lindmark et al. (1983) J. Immunol. ©0 Meth. 62:1-13. The solid phase of the immobilized protein A is preferably a column comprising a glass or a ruthenium dioxide surface, more preferably a controlled porous glass column or a ruthenium column. In some applications, the tubing has been coated with a reagent such as glycerin in an attempt to prevent non-specific adhesion of contaminants. As a first step of purification, the cell culture-derived preparation as described above is applied to a solid phase immobilized with protein A to specifically bind the antibody of interest to protein A. The solid phase is then washed to remove the > dyeable material that is non-specifically bound to the solid phase. Finally, the antibody of interest is recovered by solid phase separation by lysis. 0 b. Generation of anti-parents using eukaryotic host cells: The two vector components typically include, but are not limited to, one or more of the following: sequence L, origin of replication, one or more marker genes, enhancement Sub-elements, promoters, and transcription termination sequences. Signal sequence components Vectors for use in eukaryotic host cells may also contain a signal sequence or other 142552.doc-82-201011047 polypeptide having a specific cleavage site at the N-terminus of the mature protein or polymorphism. The heterologous signal sequence selected is preferably a signal sequence that is recognized by the host cell and processed (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signal sequences as well as viral secretion leader sequences (e. g., herpes simplex gD signal sequences) are available. The DNA of the precursor region is ligated to the coding antibody 2DNa in the reading frame. Origin of replication Generally, mammalian expression vectors do not require replication of the starting component. For example, the SV40 origin can generally be used simply because it contains early-stage (Ui) selection gene component expression and the selection vector can contain a selection gene, also known as a selectable marker. A typical selection gene encodes a protein that has the following effects: (to confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, methotrexate or tetracycline); (b) to supplement auxotrophy deficiency (related) (or) a key nutrient that is not available from the complex medium.

One example of a selection protocol utilizes drugs to block the growth of host cells. The cells that have been successfully transformed by the heterologous gene produce a protein that confers drug resistance and thus survives the selection protocol. Examples of this dominant selection use the drugs neomycin, mycophenolate, and hygromycin. Another example of a suitable selectable marker for a mammalian cell is such that it recognizes a marker capable of absorbing cellular components of the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein "and _π (preferably Long-class animal metallothionein gene), adenosine deaminase, ornithine decarboxylase, etc. For example, 'first by competing with methylamine (Mtx) (DHFR competition 142552.doc -83· 201011047 All transformants are cultured in a medium that recognizes cells transformed with the DHFR selection gene. When wild-type DHFR is used, the appropriate host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (eg, ATCC CRL- 9096) Alternatively, the DNA sequence encoding the antibody can be selected by cell growth in a medium containing a selectable marker such as an aglycoside antibiotic such as kanamycin, neomycin or G418. , wild-type DHFR protein and another selectable marker (such as aminoglycoside 3·-phosphotransferase (ΑΡΗ)) transformed or co-transformed host cells (especially wild-type sinks containing endogenous DHFR) See also U.S. Patent No. 4,965,199. (iv) Promoter component expression and selection vectors typically contain a promoter that is recognized by the host organism and operably linked to the antibody polypeptide nucleic acid. Promoter sequences of eukaryotic organisms are known. In fact, all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another one found 70 to 80 bases upstream from the start of transcription of many genes The sequence is the CNCAAT region, wherein N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be used to add a poly A tail to the coding sequence. Signals at the 3' end. All of these sequences are suitable for insertion into eukaryotic expression vectors. Antibody polypeptides from vectors in mammalian host cells are transcribed (eg) from viruses (such as polyoma, avian, viral, adenovirus) (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, giant cell virus, retrovirus, hepatitis B virus and prion 40 (SV40)) genome, from heterologous breastfeeding 142552.doc -84- 201011047 Animals A promoter (such as an actin promoter or an immunoglobulin promoter) is controlled by a promoter obtained from a heat shock promoter, the restriction being that the promoter is compatible with the host cell system. Early and late initiation of the SV40 virus The subfamily is conveniently obtained as an SV40 restriction fragment that also contains an SV4 prion origin of replication. The immediate early promoter of the human cell megavirus is conveniently obtained as a Hindlll E restriction fragment. U.S. Patent No. 4,419,446 In mammalian hosts, ▲ wins the system of expressing DNA using bovine papilloma virus as a vector. Modifications to the system are described in U.S. Patent No. 4,601,978. Alternatively, the long terminal repeat of the Rous Sarcoma Virus can be used as a promoter. ' (W enhancer element component is transcribed from high-grade eukaryotic cells to encode the DNA of the antibody polypeptide of the present invention, which is usually enhanced by inserting an enhancer sequence into a vector. It is currently known from mammalian genes (globulin, elastase, Many enhancer sequences of albumin, heart protein, and insulin. However, enhancers from eukaryotic viruses will generally be used. Examples include the Sv4 〇 enhancer (bp 1 00-270) located on the closest side of the origin of replication, The cell giant virus early promoter enhancer, the polyoma enhancer located on the nearest side of the replication origin, and the adenovirus enhancer. See also Yaniv, Nature 297:1 Τ-ΐ 8 (1982) for enhancing elements of the activated eukaryotic promoter. Although the enhancer can be spliced into the vector at position 5' or 3' of the sequence encoding the antibody polypeptide, it is preferably located at position 5 of the promoter. (W) Transcription termination component for eukaryotic The expression vector in the host cell will also generally contain sequences necessary for termination of transcription 142552.doc • 85 - 201011047 and for stable mRNA. These sequences are usually 5' and even from eukaryotic or viral DNA or cDNA. Obtained from the untranslated region of 3'. These regions contain nucleotide segments transcribed into polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. A suitable transcription termination component is bovine growth hormone. Polyadenylation region. See WO94/11026 and the expression vectors disclosed therein. (vii) Selection and transformation of host cells Suitable host cells for the selection or expression of DNA in the vectors herein include the higher eukaryotes described herein. Cells, including vertebrate host cells. The propagation of vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of suitable mammalian host cell lines are monkeys transformed by SV40 (COS_7, ATCC CRL 165 1). Kidney CV1 strain; human embryonic kidney strain (293 or 293 cells that have been subcultured in suspension culture, Graham et al, J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells ( BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216

(1980)); mouse sertoli cell (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cell (CV1 ATCC CCL 70); African green wolf kidney African green monkey kidney cell (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells ( Buffalo rat liver cell) (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC 142552.doc -86 - 201011047 CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver cancer strains (Hep G2). The host cell is transformed with the above-described expression or selection vector for producing the antibody, and cultured in a conventional medium modified as appropriate to induce a promoter, select a transformant or amplify a gene encoding the desired sequence. (yiii) Culture of host cells Host cells for producing the antibodies of the present invention can be cultured in various media. Commercially available media (such as Ham's F10, Sigma, Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium' (DMEM) (Sigma) Both are suitable for culturing host cells. In addition, Ham et al. - Man, Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255

(1980), any of the media described in U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90/03430, WO 87/00195, or U.S. Patent No. 30,985, are available. As a medium for host cells. Any of these media may optionally be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN·TM drugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar concentration range), and glucose or Equivalent energy. Any other necessary supplements that will be of a suitable concentration known to those skilled in the art may also be included. Culture conditions (such as temperature, pH 142552.doc -87 - 201011047 values and the like) are those previously used for the host cell selected for expression and will be apparent to those skilled in the art. (h) Purification of antibodies When recombinant techniques are employed, antibodies can be produced intracellularly or directly into the culture medium. ^ The antibody is produced intracellularly as a first step, for example by centrifugation or ultra-twisting to remove microparticle fragments of the host cell or lysate. Where the antibody is secreted into the culture medium, a commercially available protein concentration filter (e.g., Amicon or Millip® Re Peiiic® ultrafiltration device) is typically first used to concentrate the supernatant from the performance systems. Protease inhibitors such as PMSF may be included in any of the previous steps to inhibit protein hydrolysis' and may include antibiotics to prevent the growth of foreign contaminants. The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of the domain of any immunoglobulin present in the antibody. Protein A can be used to purify antibodies based on human gamma, gamma 2 or gamma 4 heavy bonds (Lindmark et al, J. Immunol. Meth. 62: 1-13 (1983)). Protein g is recommended for all mouse isotypes and human gamma 3 (Guss et al, EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most typically agarose, but other matrices are available. Mechanically stable matrices such as controlled microporous glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved using agarose flow rates and processing times. In the case where the antibody comprises a CH3 domain,

Bakerbond ABXTM Resin (J. T. Baker, Phillipsburg, NJ) is suitable for purification from 142552.doc -88- 201011047. Depending on the antibody to be recovered, other techniques for protein purification, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, ceria chromatography, heparin SEPHAROSETM chromatography, anion or cation exchange, may also be utilized. Resin (such as polyaspartic acid column) chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation. After any preliminary purification step, the mixture comprising the antibody of interest and the contaminant can be subjected to a dissolution buffer having a pH between about 2.5 and 4-5, preferably at a low salt concentration (eg, about 0 to 0.25 Μ salt). Performing low pH hydrophobic interaction chromatography. Immunoconjugates The invention also provides immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising the binding agents described herein to cytotoxic agents (such as chemotherapeutic agents, drugs, growth inhibitors, Any anti-Notch 1 NRR antibody or radioisotope (ie, radioactive conjugate) of a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof).

Use of antibody-drug conjugates for local delivery of cytotoxin or cytostatic agents, ie, the use of drugs to treat or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; Niculescu- Duvaz and Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Patent No. 4,975,278) which allows targeted delivery of a drug moiety to a tumor and its accumulation in cells, wherein the unconjugated 0 agent Systemic administration can result in an unacceptable degree of toxicity to normal cells and tumor cells to be sought for elimination (Baldwin et al., (1986) Lancet ρρ. (March 15, 1986): 603-05; Thorpe , 142552.doc -89- 201011047 (1985) [Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review] on Monoclonal Antibodies '84:

Biological And Clinical Applications, A. Pinchera et al. (eds., pp. 475-506). Therefore, seek the maximum efficacy with minimal toxicity. It has been reported that multiple antibodies and monoclonal antibodies are suitable for such strategies (Rowland et al. (1986) Cancer Immunol. Immunother., 21: 183-87). The drugs used in these methods include daunorubicin, amylin, amidoxime and vindesine (Rowland et al. (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin; phytotoxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al. (2000) Jour, of the Nat. Cancer Inst. 92(19): 1573-1581; Mandler et al., (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al., (2002) Bioconjugate Chem. 13 :786-791), maytansinoid (EP 1391213; Liu et al, (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623) and calicheamicin (Lode et al., (1998) Cancer Res. 58:2928; Hinman et al, (1993) Cancer Res. 53:3336-3342). These toxins can achieve their cytotoxic and cytostatic effects by a mechanism including microtubule binding, DNA binding or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when combined with large antibody or protein receptor ligands. ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate that binds to CD20 antigen by sulfur 142552.doc -90- 201011047 urea linker-chelator Murine IgG1K monoclonal antibody (discovered on the surface of normal and malignant B lymphocytes) and mIn or 9QY radioisotope composition (Wiseman et al., (2000) Eur. Jour. Nucl. Med. 27(7): 766- 77; Wiseman et al., (2002) Blood 99(12): 4336-42; Witzig et al., (2002) J. Clin. Oncol. 20(10): 2453-63; Witzig et al., (2002) J_Clin Oncol. 20(15): 3262-69). Although ZEVALIN is active against B-cell non-Hodgkin's Lymphoma (NHL), administration of the drug causes severe and long-term hematocytopenia in most patients. MYLOTARGTM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug complex consisting of hu CD33 antibody linked to calicheamicin, was approved for injection in 2000. Treatment of acute myeloid leukemia. (Drugs of the Future (2000) 25 (7): 686; US Patent 4, 970, 198

No. 5,079,233, 5,585,089, 5,606,040, 5,6937,62, 5,739,116, 5,767,285, 5,773,001). Cantuzumab mertansine, Immunogen, Inc., an antibody drug conjugate consisting of a huC242 antibody linked to a maytansinoid drug moiety (DM1) via a disulfide linker SPP, is developing To Phase II trials for the treatment of cancers that exhibit CanAg, such as colon cancer, pancreatic cancer, stomach cancer, and other cancers. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.), an antibody drug conjugate consisting of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to a maytansinoid drug moiety (DM1), positive It is under development for the potential treatment of prostate tumors. Auristatin peptide, Ollis lamp E (AE) and 142552.doc -91 - 201011047 monomethylauristatin (MMAE) (synthetic analog of sea rabbit toxin) line and embedded The monoclonal antibody cBR96 (specific for Lewis Y on cancer) and cACIO (specific for CD30 on hematological malignancies) bind (Doronina et al. (2003) Nature Biotechnology 21 (7): 778-784 And is in the process of treatment development. Chemotherapeutic agents suitable for use in generating immunoconjugates are described herein (e.g., above). The enzymatically active toxins and fragments thereof which can be used include: diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa (Psewc/oAnowa®, marigold toxin A, acacia toxin) Abrin) A bond, modeccin A bond, alpha-sarcin, tung tree (J/ewr/ies protein, carnation protein, foreign commercial land)

Awericawa) protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, paradise fruit protein Gelonin), mitogen (mitogellin), restrictocin, phenomycin, enomycin, and trichothecene. See, for example, WO 93/21232, published October 28, 1993. Various radionuclides can be used to generate radiolabeled antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. A combination of an antibody and a cytotoxic agent is prepared using a plurality of bifunctional protein couplers such as N-succinimide-3-(2-pyridyldithiol) propionate ( SPDP), iminothiolane (IT), bifunctional derivatives of sulfhydryl esters (such as dimethyl dimethyl imidate hydrochloride), active esters (such as diammonium suberate)醯imino ester), aldehydes (such as glutaraldehyde), 142552.doc •92· 201011047 bis-azido compounds (such as bis-(p-azidobenzylidene) hexamethylenediamine), bis-diazo Salt derivatives (such as bis(p-diazobenzyl)-ethylenediamine), diisocyanates (such as toluene 2,6. diisocyanate) and bis-active fluorine compounds (such as 1,5-fluoro-2) , 4-nitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987). The carbon-14-labeled 1 isothiocyanatobenzyl-3-methyl-2-ethylidene pentaacetic acid pentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding a radioactive nucleotide to an antibody. See WO94/11026. An antibody with one or more small molecule toxins (such as calicheamicin, maytansinoid, dolphin toxin, orlistin, trichothecene steroid toxin, and CC1065) and derivatives thereof having toxin activity The combination is also covered herein. i. Maytansine and Maytansinoid In some embodiments, the immunoconjugate comprises an antibody (full length or fragment) of the invention that binds to one or more maytansinoid molecules.

Maytansin is a mitotic inhibitor that acts by inhibiting tubulin polymerization. Maytansin was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Later, certain microorganisms were also found to produce maytansinoids, such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,151,042). For example, U.S. Patent Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, No. 4,315,929, 142,552.doc-93-201011047 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533 Synthetic maytansinol and its derivatives and analogs are disclosed. The maytansinoid moiety is an attractive drug moiety in an antibody drug conjugate because it is: (i) relatively easy to prepare by fermentation or chemical modification or derivatization of the fermentation product, (ii) suitable Derivatization of a functional group suitable for binding to an antibody via a non-disulfide bond linker '(Hi) is stable in plasma, and (iv) is effective against a plurality of tumor cell lines. An immunoconjugate comprising a maytansinoid, a method of making the same, and a therapeutic use thereof are disclosed, for example, in U.S. Patent Nos. 5,2,8, 〇20, 5,416,064 and European Patent EP 〇425 235 B1. The disclosure is expressly incorporated herein by reference. Liu et al, Proc. Natl. Acad. Sci. USA 918618-8623 (1996) describe immunoconjugates comprising maytansine (designated DM1) linked to monoclonal antibody C242 against human colorectal cancer. The conjugate was found to be highly cytotoxic to the cultured colon cancer cells and exhibited anti-tumor activity in an in vivo tumor growth assay. Chari et al, Cancer Research 52: 127-131 (1992) describe the binding of maytansinoid to a class I antibody A7 that binds to an antigen on a human colon cancer cell line via a disulfide bond linker or to a HER_2/neu oncogene. Another gas-type single-body k-body ΤΑ. 1 combined immunoconjugate. To test the cytotoxicity of the τα丨_maytansin conjugate on human breast cancer cell line SK-BR-3 (each of which exhibits 3 X 1 HER HER-2 surface antigen) in vitro. A similar degree of cytotoxicity of the maytansinoid drug can be increased by increasing the number of maytansine molecules per antibody molecule by increasing H2552.doc-94-201011047. The A7_classendensin conjugate exhibits low systemic cytotoxicity in mice. The antibody-maytansin conjugate is prepared by chemically linking an antibody to a maytansinoid molecule without significantly reducing the biological activity of the antibody or maytansinoid molecule. See, for example, U.S. Patent No. 5,2,8, 〇 2 ( (the disclosure of which is incorporated herein by reference). Each antibody molecule binds an average of 3 to 4 maytansinoid molecules that have been shown to enhance cytotoxicity against the target cells. The effect is not negatively affected by the antibody's Wei or solubility, but even one molecule of toxin/antibody is expected It enhances cytotoxicity and is superior to the use of naked antibodies. Maytansin is well known in the art and can be synthesized by known techniques or isolated from natural sources. Appropriate mayonoids are disclosed, for example, in the United States • Patent No. 5, 2G8, G2 Continuation and Reading and other specific disclosures. Preferred maytansinoids are maytansinol and modified maytansinoids, such as various maytansinol esters, modified at the aromatic ring of the maytansinol group or other positions. A variety of linking groups are known in the art for the manufacture of antibody-type maytansin conjugates, including, for example, U.S. Patent No. 5,2,8,020 or European Patent 0 425 235 Bl^Chari^A &gt; Cancer Research U.S. Patent Application Serial No. </RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> </ RTI> <RTIgt; And other linking groups. The inclusion of the linker component sMc (the antibody-maytansin conjugate) can be prepared as disclosed in U.S. Patent Application Serial No. 1/960,602, the entire disclosure of which is incorporated by reference. a disulfide group, a sulfur group, an acid labile group, a photolabile group, a peptidase labile group or a cool enzyme labile group disclosed in the patent, wherein the disulfide 142552.doc • 95· 201011047 The base and thioether group are preferred. Other linkers are described and exemplified herein. The combination of an antibody and a maytansinoid can be made using a variety of bifunctional protein couplers, such bifunctional protein couples. The mixture is, for example, N_amber quinone imino 3-(2-pyridyldithio)propionate (SPDP), amber quinone imine _4_(]^_cis-butyl di-iminomethyl a cyclohexyl phthalate (SMCC), an imidothiolane (IT), a difunctional derivative of a quinone imide (such as dimethyl dimethyl imidate hydrochloride), an active ester (such as disuccinimide suberate), complexes (such as valeric acid), bis-azido compounds (such as bis(p-azidobenzyl hydrazine) Hexamethylenediamine), bis-diazonium salt derivatives (such as bis(p-diazonium bromide)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-Difluoro-2,4-dinitrobenzene. Particularly preferred coupling agents include N-succinimide _3_(2-pyridyldithio)propionate (SPDP) (Carlsson et al. Human, Biochem. J. 173: 723-737 (1978)) and N-succinyl imino-4-(2-pyridylthio)pentanoate (spp) to provide a disulfide bond. The linker can be linked to the maytansinoid molecule at various positions. For example, a conventional coupling technique can be used to form an ester bond by reacting with a hydroxyl group. The reaction can occur at the c_3 position having a hydroxyl group, via hydroxy group. The methyl-modified C-14 position, the hydroxyl-modified c-15 position, and the C-20 position having a hydroxyl group. In a preferred embodiment, the C-3 position of the maytansinol or maytansinoid analog is formed. Bonding. U. Orlistin and Dolphin Toxins In certain embodiments, the immunoconjugate comprises an ornithine-like analog and a derivative of the dolastatin The antibody of the present invention (US 242552.doc • 96-201011047, Nos. 5, 635, 483 and 5, 780, 588) has shown that the rabbit toxin and orlistin interfere with microtubule dynamics, GTP hydrolysis and nuclear and cell separation (Woyke et al. Human (2001) Antimicrob·Agents and Chemother. 45(12): 3580-3584) and has anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., (1998) Antimicrob·'Agents Chemother. 42: 2961-2965). The dolastatin or orlistin drug moiety can be attached to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).

Illustrative orlistin embodiments include N-terminally linked monomethyl orlistin disclosed in Monomethylvaline Compounds Capable of Conjugation to Ligands, U.S. Patent Application Serial No. 10/983,340, filed on Nov. 5, 2004. The drug portions DE and DF, the disclosures of which are expressly incorporated herein by reference in their entirety. Peptidyl drug moieties can generally be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schr6der and K. Ltibke, [The Peptides], Vol. 1, pp. 76-136, 1965, Academic Press). The Ollistin/Dolphin toxin drug portion can be based on U.S. Patent Nos. 5,635,483 and 5,780,588; Pettit et al., (1989)·!· Am. Chem. Soc. 111:5463-5465; Pettit et al., (1998) ) 11 to - Cancer Drug Design 13: 243-277; Pettit, GR et al, Synthesis, 1996, 719-725 and Pettit et al, (1996) J. Chem. Soc. Perkin Trans. 1 5: 859-863 The method is to prepare. See also October 27, 2005 曰 published by Doronina (2003) Nat. Biotechnol. 21(7): 778-784; [Monomethylvaline Compounds Capable of 142552.doc -97- 201011047

Conjugation to Ligands, US20050238649, which is incorporated herein by reference in its entirety, for example, the disclosure of the &lt;RTIgt;&lt;/RTI&gt; </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt;

Hi, calicheamicin In other embodiments, the immunoconjugate comprises an antibody of the invention that binds to one or more calicheamicin molecules. The coccimycin family of antibiotics is capable of producing double-strand DNA breaks at a sub-picumer concentration. For the preparation of the conjugates of the calicheamicin family, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296 (all patents) All belong to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, gamma 1, 1, 21, alpha, N-ethylidene tau/, PSAG, and e^Hinman et al, Cancer Research 53: 3336-3342 ( 1993), Lode et al., Cancer Research 58: 2925-2928 (1998) and the aforementioned U.S. patent to American Cyanamid). Another anti-tumor drug that the antibody can bind to is QFA, which is an anti-folate agent. Both lutein and QFA have intracellular sites of action and are not susceptible to crossing the plasma membrane. Thus, antibody-mediated internalization of cells absorbing these agents greatly enhances their cytotoxic effects. Iv. Other cytotoxic agents Other anti-tumor agents that can be combined with the antibodies of the invention include BCNU, streptozoicin, vincristine, and 5-fluorouracil, as described in U.S. Patent Nos. 5,053,394 and 5,770,710. A family of agents of the ll_E33288 complex and Esperazine (U.S. Patent No. 142,552.doc-98-201011047, 5,877,296). Enzymatically active toxins and fragments thereof which may be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin octapeptide (from Pseudomonas aeruginosa), ricin A chain, abrin toxin a chain, Modizin a Chain, α-pyromycin, tung tree protein, carnation protein, foreign commercial protein (pApi, pApu and ρΑρ_ S), bitter melon inhibitor, hema protein, croton toxin, sage inhibitor, white matter, mitosis , limited, bacteriocin, phenolic acid, enamel

And mycotoxins. See, for example, W〇 93/21232, published on October 28, 1993. The invention further encompasses an immunoconjugate formed between an antibody and a compound having nuclear degrading activity, such as a ribonuclease or a DNA endonuclease such as deoxyribonucleic acid 6# (DNase). To selectively destroy a tumor, the antibody can comprise a highly radioactive atom. A variety of radioisotopes are available for the production of radiolabeled antibodies. Examples include At2&quot;, I(1), ι125, γ9〇, ReI86, 188, sml53, 212, p32,

Pb and its radioisotope. When the conjugate is used for detection, it may contain radioactive atoms for scintigraphy studies (eg, 9^*.23) or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, melon Spin label, such as iodine-123 (again), iodine-131, indium_1U, fluorine_19, carbon-13, nitrogen-15, oxygen-17, chaos, violent or iron. Radioactivity can be known in a known manner A label or other label is incorporated into the conjugate. For example, the peptide can be synthesized biologically, or can be synthesized by chemical amino acid synthesis using a suitable amino acid precursor (eg, comprising fluorine instead of hydrogen) Peptides such as Tc99m or P23, Re 86, Rei88 and Inm can be linked by a cysteine residue in the peptide via 142552.doc • 99- 201011047. 钇-90 can be linked via an lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) was used to incorporate moth-123. [Monoclonal Antibodies in Immuno sc intigraphy] (Chat al, CRC Press 1989) Other methods. Combinations of antibodies and cytotoxic agents can use a variety of bifunctional protein coupling agents. The bifunctional protein coupling agents are, for example, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), amber quinone-4-(N-cis-butene) A difunctional derivative of an enediminoimidomethyl)cyclohexane-1-carboxylate (SMCC), an imidothiolane (IT), or a quinone imide (such as hexamethylenediamine) Dioxalate hydrochloride), active esters (such as disuccinimide suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzylhydrazine) Hexamethylenediamine), bis-diazonium salt derivatives (such as bis-(p-diazonium bromide)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and double active fluorine Compounds such as 1,5-difluoro-2,4-dinitrobenzene. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylidamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radionucleotides to antibodies. See WO94/ 11026. The linker can help to fine cells a "cleavable linker" released by a toxin drug. For example, an acid labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing link may be used. (Chari et al. 'Clinic Research 52: 127-131 (1992); U.S. Patent No. 5,208,020). 142552.doc -100- 201011047 The compounds of the present invention expressly encompass, but are not limited to, the incorporation of the following commercially available cross-linking reagents (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA) into the 0 (:: 3 PCT? 8, £]\408, 〇]^68, 1^¥8,: 1: (:-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfonic acid-EMCS, sulfonate-GMBS, sulfonate-KMUS, sulfonate-MBS, sulfonate-SIAB, sulfonate-SMCC and sulfonate-SMPB and SVSB (amber quinone-amino-(4-vinyl sulfone) Benzoate) See pages 467-498, 2003-2004 Applications Handbook and mm ww Catalog. v. Preparation of antibody drug conjugates

In the antibody drug conjugate (ADC) of the invention, the antibody (Ab) binds to one or more drug moieties (D) via a linker (L), e.g., from about 1 to about 20 drug moieties per antibody. The ADC of Formula I can be prepared by a number of routes using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) nucleophilic groups of antibodies and bivalent linker reagents via covalent bond reactions To form Ab-L, which in turn reacts with drug moiety D; and (2) the nucleophilic group of the drug moiety reacts with the divalent linker reagent via a covalent bond to form DL, which in turn reacts with the nucleophilic group of the antibody . Other methods of preparing ADCs are described herein.

The Ab-(L-D)p I linker can be composed of one or more linker components. Exemplary linker components include 6-maleimide hexamethylene fluorenyl ("MC"), maleimide propyl thiol ("MP"), leucine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl 142552.doc -101 - 201011047 (pAB"), 4-(2-pyridylthio)penta Acid N-succinimide g ("SPP"), 4-(N-methylene-2-imidazolylmethyl)cyclohexane-indole-formic acid N-coded imidate (r SMCC) And (4_iodo-ethinyl)aminobenzoic acid N-glass-baked imine ester ("SIAB" p additional linker component is known in the art and some are described herein. See [Monomethylvaline Compounds Capable of Conjugation to [5]

Ligands, U.S. Patent Serial No. 10/983,34, the entire contents of which is incorporated herein by reference. In some embodiments, the linker can comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides or pentapeptides. Exemplary dipeptides include: phytic acid-citrulline (vc4 val_cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-glycine-glycine (gly-val-cit) and glycine-glycine-glycine (giy_gly_gly). Amino acid residues comprising an amino acid linker component include naturally occurring amino acids and minor amino acids and non-naturally occurring amino acid analogs such as citrulline. The amino acid linker component can be designed and optimized for its selectivity for enzymatic cleavage of specific enzymes such as tumor-associated proteases, cathepsins B, c and D or plasminogenase. Nucleophilic groups on the antibody include, but are not limited to, (i) an N-terminal amine group; (ii) a side chain amine group, such as an amine acid; (a) a side chain thiol group, such as cysteine And (IV) a saccharide hydroxyl or amine group at which the antibody is glycosylated. An amine, a thiol, and a nucleophilic group are capable of reacting with an electrophilic group on a linker moiety and a linker reagent comprising the following: to form a covalent bond: (an active ester such as an NHS ester, HOBt ester, haloformic acid 142552.doc •102· 201011047 vinegar and acid halide; (ii) alkyl and nodal dentate, such as _acetamide; (iii) conjugate, ketone, carboxyl and cis-butene Diterpenoid imine group. Some antibodies have a reducible interchain disulfide bond, ie a cysteine bridge. The antibody can be treated by treatment with a reducing agent such as DTT (dithiothreitol). The reactivity of the linker reagents is combined. Therefore, theoretically each cysteine bridge will form two reactive thiol nucleophiles. This can be achieved by making the lysine and the 2-iminothiolane (Traut reagent). The reaction converts the amine to thiol to introduce additional pro-nuclear groups into the antibody by introducing one, two, three, four or four or more cysteine residues Introducing a reactive thiol group into an antibody (or a fragment thereof) (eg, preparing one or more non-natural cysteamine amines) Mutant antibody of a base acid residue) The antibody drug conjugate of the present invention can also be produced by modifying an antibody to introduce an electrophilic moiety reactive with a nucleophilic substituent on a linker reagent or drug. The sugar of the antibody of the antibody is oxidized by, for example, a periodate oxidizing reagent to form an aldehyde group or a ketone group which can react with the amine group of the linker reagent or the drug moiety. The obtained imine Scheiff base group can be Forming a stable bond, or may be reduced, for example, by a borohydride reagent to form a stable amine bond. In one embodiment, the carbohydrate moiety of the glycosylated antibody is reacted with galactose oxidase or sodium metaperiodate. Producing a green group (Hermans〇n, Bi〇conjUgate Techniques) in the protein that reacts with a suitable group on the drug. In another embodiment, it contains a \terminal leucine or a sulphate The protein of the amino acid residue can be reacted with sodium metaperiodate to produce an aldehyde instead of the I* group acid (Geoghegan &amp; Stroh, (1992) Bioconjugate Chem. 3: 138-146; U.S. Patent No. 5,362,852). The aldehyde can be combined with the drug moiety 142552. Doc •103- 201011047 or linker nucleophile reaction. Similarly, nucleophilic groups on the drug moiety include, but are not limited to: amines, thiols, hydroxyls, hydrazines, hydrazines, hydrazines, thioscarbazide, An oxime carboxylate group and an aryl sulfonium group capable of reacting with a linker moiety and an electrophilic group on a linker reagent comprising the following: to form a covalent bond: (1) an active ester such as an NHS ester, HOBt esters, tooth-formates and acid-based compounds; (Η) alkyl and benzyl halides, such as haloacetamide; (iii) aldehydes, ketones, carboxyl groups and maleimide groups . Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be produced, for example, by a recombinant technique or peptide synthesis. The length of the DNA may comprise separate regions encoding two portions of the conjugate that are adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired conjugate properties. In yet another embodiment, the antibody can be combined with a "receptor" (anti-streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the individual, followed by a scavenger The unbound conjugate is removed from the circulation and subsequently administered a "ligand" (eg, avidin) that binds to a cytotoxic agent (eg, a radionucleotide). A pharmaceutical formulation by using an antibody of the desired purity with a physiologically acceptable optional excipient or stabilizer; Kunhe (Heart Calendar ~ (4) (10) kg (10) · Heart / PW defeat; ^ 20 version ( 2_)) An analgesic formulation comprising an antibody of the invention is prepared in the form of an aqueous solution, an East Dry Flavor or other dry formulation for feeding. At the dosages and concentrations employed, acceptable carriers, excipients, or stabilizers are non-toxic to the recipient, and include buffering agents, such as 142552.doc -104- 201011047 such as phosphates, citrates, histidines And other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as gasified octadecyldimethylbenzylammonium; gasified hexahydrocarbon quaternary ammonium (hexameth〇nium chi〇ride) Benzalkonium chloride;

(benzethonium chloride); phenol, butanol or stilbene; alkyl parabens such as methylparaben or propylparaben; catechol, resorcinol; cyclohexanol; _ pentanol and m-cresol "low molecular weight (less than about 1 residue) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid , such as glycine, acetophenone, aspartame, histidine 'arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, Such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants Such as TWEENTM, PLUR0NICSni or polyethylene glycol (PEG). As required for the particular indication being treated, the formulations herein may also contain more than one active compound, preferably having complementary activities that do not adversely affect each other. Active compound Suitably in combination in an amount effective to achieve the intended purpose. The active ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymercapto cellulose or gelatin microcapsules and poly( In decyl methacrylate microcapsules, embedded in a gelatinous drug delivery system (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or embedded in a macroemulsion. Other technologies are revealed at 142552.doc •105- 201011047

Remington: The Science and Practice of Pharmacy% IQ Edition (2000). Formulations to be administered in vivo must be sterile. This is easily accomplished by filtration through a sterile filter. A sustained release preparation can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing immunoglobulins of the invention. The matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include poly Ester, hydrogel (for example, poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol polylactic acid vinegar (US Patent No. 3,773,919), L-glutamic acid and γ-ethyl-L - a copolymer of lysine, a non-degradable ethylene-vinyl acetate, a degradable lactic acid copolymer, such as LUPRON DEPOTTM, consisting of a lactic acid-glycolic acid copolymer and leuprolide acetate. Injection of microspheres) and poly-D_(a)_3_-butyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 1 day, some hydrogel-releasing proteins last a short time. When the encapsulated immunoglobulin is maintained in the body for a long time, it may be denatured or aggregated due to exposure to water, resulting in loss of biological activity and possible changes in immunogenicity. Sight Depending on the mechanism involved, a reasonable stabilization strategy can be designed. For example, if the aggregation mechanism is found to be formed by the intermolecular s_s bond of the thiodisulfide interchange, the residue can be modified from the acidic solution. Stabilizing the composition by controlling the moisture content, using appropriate additives, and developing a polymer-based composition. III. Therapeutic Use 142552.doc -106· 201011047 Antibodies and antibodies that bind to both HER2 and VEGF as described herein Fragments (eg, 'bHl-44 or bHl-88 or fragments thereof) can be used to treat tumors (including precancerous, non-metastatic and cancerous tumors (eg, early cancer)), for the treatment of autoimmune diseases, for treatment Administering a condition, for treating a disease involving abnormal activation of HER2 or for treating a cancer having development (for example, breast cancer, colorectal cancer, lung cancer 'renal cell carcinoma' neurogenic kenoma or nest cancer), jk tube formation disorder An individual, an autoimmune disease, or an individual involved in the risk of a disease that is abnormally activated by HER2. The term cancer encompasses a collection of proliferative disorders including, but not limited to, precancerous growth, good Tumors and malignant tumors. Benign tumors remain at the initial site and do not have the ability to infiltrate, invade, or metastasize to distant sites. Malignant tumors • Tumors will invade and damage other tissues around them. - the ability of the site to spread to other parts of the body (metastasis), usually via the bloodstream or via the lymph nodes located in the lymph nodes. Classification of the primary tumor by the type of tissue from which the primary tumor is produced; by causing cancer cells Tissue type classifies metastatic tumors. Malignant tumor cells become more abnormal over time and appear to be less like normal cells. This change in the appearance of cancer cells is called tumor grade, and cancer cell lines are described as fully differentiated, moderately differentiated, and poorly Differentiated or undifferentiated. The well-differentiated cells look quite normal and resemble the normal cells from which they are produced. Undifferentiated cells are cells that have become abnormal so that it is no longer possible to determine the origin of the cell. The tumor can be a solid tumor or a non-solid tumor or a soft tissue tumor. Examples of soft tissue tumors include leukemia (eg, chronic myelogenous leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid white 142552.doc • 107- 201011047 blood disease mature B cell acute lymphoblastic leukemia, chronic lymphocytes Leukemia, anterior lymphocytic leukemia or hairy cell leukemia) or lymphoma (eg, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include cancer of any body tissue other than blood, bone marrow or lymphatic system. Solid tumors can be further divided into solid tumors of epithelial origin and solid tumors of non-epithelial origin. Examples of epithelial solid tumors include the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, mouth, stomach, duodenum, small intestine, large intestine, anus, gallbladder, lip, nasopharynx, Tumors of the skin, uterus, male genitalia, urinary tract, bladder and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. Epithelial cancer usually develops from a benign tumor to a pre-invasive stage (e.g., a primary cancer), a malignant cancer that has penetrated the basement membrane and invades the epithelial cell matrix. Multispecific antibodies that bind to both VEGF and HER2 (e.g., &lt;RTIgt;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos;&apos; It has been well established that angiogenesis is involved in the pathogenesis of a variety of conditions. Such conditions include solid tumors and metastases, atherosclerosis, post-lens fibrous tissue hyperplasia, hemangioma, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathy (eg, diabetic retinopathy), age-related macular degeneration (AMD), neovascular glaucoma), immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis and psoriasis. Folkman et al, J. Biol. Chem., 267: 10931-10934 (1992); 142552.doc • 108 · 201011047

Klagsbrun et al., Annu. Rev. Physiol. 53:217-239 (1991); and Garner A., [Vascular diseases] in Pathobiology of 〇cular Disease. A Dynamic Approach, Garner A., Klintworth GK, ed. 2nd edition (Marcel Dekker, NY, 1994), pp. 1625-1710. Abnormal angiogenesis occurs when new circulatory, inadequate, or inappropriate (e.g., from a medical point of view, where the location, timing, or episode of angiogenesis is growing) grows in a diseased state or causes it to cause a disease state. Excessive, inappropriate, or uncontrolled angiogenesis occurs when promoting the progression of a diseased state or the growth of new blood vessels that cause disease, such as cancer (especially vascularized solid tumors and metastatic tumors (including colon, lung cancer (especially For small cell lung cancer) or prostate cancer)), diseases caused by new blood vessels in ophthalmology (especially for diabetic nephropathy, retinopathy, early diabetic retinopathy or age-related macular degeneration (AMD)), diabetic macular edema Cerebral edema (eg, with acute stroke/locking head injury/trauma), synovitis, vasospasm in rheumatoid arthritis, ossifying myositis, hypertrophic bone formation, refractory abdomen water Symptoms, polycystic ovarian disease, the third space of fluid diseases (pancreatitis, chamber syndrome, burns, bowel disease), uterine fibroids, premature babies, eye horns Jin Jin (Iris redness), malignant hydronephrosis, blood vessels Restenosis, hemangioblastoma (such as hemangioma); inflammatory renal disease (such as spheroid nephritis, especially mesangial proliferative spheroid nephritis), dissolution Uremia syndrome, diabetic nephropathy or blood pressure nephrosis, various inflammatory diseases (such as arthritis (especially rheumatoid arthritis), inflammatory bowel disease, psoriasis, psoriasis (· 生生知炎,斑斑) Block psoriasis, sarcoidosis, arteriosclerosis and diseases after transplantation), renal allograft rejection, endometriosis or 142552.doc 201011047 Ling asthma and other conditions of 7G or more. New blood vessels can be raised In diseased tissues, poor normal tissues, and in the case of cancer, new blood vessels can cause tumor cells to escape to the circulation and remain in other organs (tumor metastasis). Insufficient angiogenesis occurs in the presence of disease-promoting conditions. Deteriorating inappropriate blood vessels grow, for example, in diseases such as coronary artery disease, stroke, and delayed wound healing. In addition, ulcers, strokes, and heart attacks can be caused by the absence of natural recovery. The invention encompasses the use of antibodies (eg, bm_81 or bm_44 antibodies) that specifically bind both VEGF and HER2. Those patients at risk of suffering from such diseases. Other patients who are candidates for receiving the compositions of the present invention are at risk of or suffering from the following diseases: abnormal proliferation of fibrous tubule tissue, acne, acquired immunodeficiency syndrome Arterial obstruction, atopic keratitis, 'Tianzhushengsheng' Bayer, Behcet's disease, blood-borne tumor, carotid artery obstructive disease, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic grape Membrane inflammation, chronic vitreitis, contact lens hyperopia, corneal transplant rejection, corneal neovascularization, corneal neovascularization, Crohn's disease, Eales disease, epidemic keratoconjunctivitis, fungal ulcer, Herpes simplex infection, herpes zoster infection, hyperviscosity syndrome, Kaposi's sarcoma, leukemia, lipid degradation, Lyme's disease, marginal stratum corneum separation, Mooren ulcer In addition to leprosy, Myc〇bacteria infection, myopia, ophthalmic neovascular disease, small optic disc , 〇sler_Weber syndrome (Osler-Weber-Rendu), osteoarthritis, Paget's disease, flat 142552.doc 110 201011047 inflammation, pemphigoid, blisteric keratoconus, polyarteritis, post-laser complications , protozoal infection, elastic pseudo-xanthoma, pterygium dry keratitis, radial keratotomy, retinal neovascularization, retinopathy of prematurity, posterior fibrous hyperplasia, sarcoidosis, scleritis, sickle cell anemia , S〇grenis syndr〇me, solid tumor, Stargartis disease, Steven, s Johnson disease, upper keratitis, syphilis, systemic Lupus, n Terrien's marginal degeneration, echinococcosis, Ewing sarc〇ma tumor, neuroblastoma tumor, osteosarcoma tumor, retinoblast Tumor tumor, rhabdomyosarcoma tumor, ulcerative colitis, venous obstruction, Vitamin A deficiency, Wegener's sarcoma disease (Wegener, s Sarcoidosis), undesired angiogenesis associated with diabetes, parasitic diseases, abnormal wound healing, surgery, injury or post-traumatic hypertrophy (eg, acute lung injury/ARDS), inhibition of hair growth, inhibition of ovulation and corpus luteum formation, inhibition of planting Into and inhibit embryo development in the uterus. Anti-angiogenic therapy is indicated for general treatment of transplant rejection, pneumonia, primary pulmonary hypertension, renal disease, initial sputum and pleural effusion, diseases and conditions characterized by adverse vascular permeability (eg 'with Edema associated with brain tumors, ascites associated with malignant diseases, Meigs syndrome, pneumonia, renal syndrome, pericardial hydrops (as associated with pericarditis), and cardiovascular disease Infiltration (such as myocardial infarction and conditions after stroke) and similar diseases) and stagnation. 142552.doc -111 · 201011047 Lymphatic disease includes angiofibroma (easy neonatal ▲ ductal glaucoma (eye swelling, other angiogenesis of the invention depends on the abnormal blood of bleeding blood vessels), growth of new tubules), arteriovenous Malformation (AVM; abnormal connection between arteries and veins

form). IV. Dosage and Formulations Antibodies (e.g., bHl-44 or bHl-81) or antibody fragment compositions will be formulated, administered, and administered in a manner consistent with good pharmaceutical practice. The factors considered herein include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual, the cause of the condition, the location of the agent, the method of administration, the schedule of administration, and other factors known to the physician. The "therapeutically effective amount" of the antibody or antibody fragment to be administered will be determined by such considerations and is the minimum amount required to prevent, ameliorate or treat the cancer or autoimmune disorder. The antibody or antibody fragment does not need (but optionally) be formulated with one or more agents to present a risk for the prevention or treatment of cancer or autoimmune disorders or the development of cancer or autoimmune disorders. The effective amount of such other agents will depend on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors discussed above. Such agents are generally used in the same dosages and routes of administration as used above or in the amounts used to date from 142552.doc • 112- 201011047 1% to 99%. Reducing or treating cancer typically involves reducing one or more symptoms or medical problems associated with cancer. A therapeutically effective amount of a drug may achieve one or a combination of the following effects: reduction (at least 丨〇%, 2%%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) %, 100% or 1〇〇〇/0 or more) the number of cancer cells; reducing or inhibiting tumor size or tumor burden; inhibiting (ie, reducing and/or stopping to some extent) cancer cells infiltrating into the peripheral device g, Reduces hormone secretion in the case of adenomas; reduces vascular density; inhibits tumor metastasis; reduces or inhibits tumor growth; and/or somewhat alleviates one or more symptoms associated with cancer. In some embodiments, the antibody or antibody fragment is used to prevent the onset or recurrence of a cancer or autoimmune disorder in an individual. In the present invention, the present invention can be used to increase the duration of survival of a human patient susceptible to cancer or autoimmune immune disease or by having a cancer or autoimmune disorder. The duration of survival is defined as the time from the first drug administration to death. The duration of survival can also be measured by the stratification risk ratio (HR) of the treatment group versus the control group, which indicates the risk of death of the patient during the treatment period. In yet another embodiment, the treatment of the present invention significantly increases the response rate of a human patient population susceptible to cancer or diagnosed with cancer treated by various anti-cancer therapies. The response rate is defined as the percentage of treated patients who respond to treatment. In one aspect, the use of an antibody or antibody fragment of the invention in combination with surgery, light therapy, or - or multiple chemotherapeutic agents significantly increases the number of treated patients compared to a group treated only by surgery, radiation therapy, or chemotherapy alone. The response rate of the group, which has a Chi-square P-value of less than 0.005 ° 142552.doc -113- 201011047 Additional measures for the therapeutic efficacy of cancer treatment are described in the U.S. Patent Application Publications In No. 20050186208. By mixing standard ingredients of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers using standard methods known in the art (Remington's Pharmaceutical Sciences (20th Edition), A Gennaro, ed., 2000, Lippincott, Williams &amp; Wilkins, Philadelphia, PA) to prepare therapeutic formulations. Acceptable carriers include physiological saline or buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin , gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, glutamine, aspartame, arginine or lysine; monosaccharide, Sugars and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants Such as TWEENTM, PLURONICS top or PEG. Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride and preferably at about physiological concentrations. The formulations of the present invention may contain a pharmaceutically acceptable preservative, as the case may be. In some embodiments, the preservative concentration ranges from 0.1% to 2.0% (typically v/v). Suitable preservatives include those known in the medical arts. Alcohol, benzophenone, m-nonylphenol, decyl p-hydroxybenzoate and propyl p-hydroxybenzoate are preferred preservatives. The formulation of the present invention may comprise a pharmaceutically acceptable surfactant at a concentration of from 0.005% to 0.02%, as the case may be. 142552.doc -114- 201011047 The formulations herein may also contain more than one active compound, preferably active compounds having complementary activities that do not adversely affect each other, as required for the particular indication being treated. The molecules are suitably present in combination in an amount effective to achieve the intended purpose.

The active ingredient may also be embedded in microcapsules prepared by, for example, coacervation techniques or by interfacial polymerization (for example, thiolated cellulose or open knee microcapsules and poly(methacrylate methacrylate) microcapsules, respectively). It is embedded in a gelatinous drug delivery system (such as liposome, albumin microspheres, microemulsion, nanoparticle and nanocapsules) or embedded in a macroemulsion. Such techniques are disclosed in the aforementioned Remingt(R), s Pharmaceutical Sciences. A performance release formulation can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(ethylene glycol)), polylactic acid vinegar (U.S. Patent No. 3,773,919) a copolymer of L-face acid and γ-ethyl-L- face vinegar, non-degradable ethylene-vinyl acetate, a degradable lactic acid-glycolic acid copolymer (such as LUPRON DEPOTtm, from lactic acid to ethanol) An injectable microsphere composed of an acid copolymer and leuprolide acetate and poly-d_(_) 3_butyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for more than 100 days', certain hydrogel-releasing proteins take a relatively short period of time. When the encapsulated antibody is maintained in the body for a long period of time, it is variably or aggregated due to exposure to moisture at 37 ° C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, 142552.doc -115- 201011047 can be designed to be a stable strategy. For example, if the aggregation mechanism is found to be formed by the intermolecular s-s bond of the thio-disulfide interchange, it can be modified by modifying the Wei group, lyophilizing from the acidic solution, controlling the moisture content, and using appropriate additives. And specific polymer matrix compositions are developed to achieve stabilization. According to known methods, such as intravenous administration in the form of a bolus, or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracranial, subcutaneous, intra-articular, intrasynovial, intrathecal, meridian Oral, topical or inhalation routes' The administration of antibodies and antibody fragments (e.g., bHl-44 or bHl-81 or fragments thereof) as described herein to a human subject. If extensive side effects or toxicity are associated with VEGF and/or HER2 antagonism, topical administration is required. Ex vivo strategies can also be used for therapeutic applications. An ex vivo strategy involves transfecting or transducing cells obtained from an individual with a polynucleotide encoding an antibody or antibody fragment. The transfected or transduced cells are then returned to the individual. The cells can be of any of a variety of types including, but not limited to, hematopoietic cells (eg, bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, Epithelial cells, endothelial cells, keratinocytes or muscle cells. In one example, an antibody (e.g., &apos;bHl-44 or bHl-81) or antibody fragment is administered topically (e.g., by direct injection) when the tumor condition or location permits, and the injection can be repeated periodically. The antibody or antibody fragment can also be delivered systemically to the individual or directly to the tumor cell, e.g., to the tumor or to the tumor bed after surgical removal of the tumor&apos; to prevent or reduce local recurrence or metastasis. 142552.doc • 116- 201011047 ν·Manufactured Articles and Kits Another embodiment of the present invention is an article of manufacture containing a substance suitable for the treatment of autoimmune diseases and cancer. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable cereals include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous infusion bag or vial having a stopper pierceable by a hypodermic needle). At least one of the active agents in the composition is a multispecific antibody or an anti-body tablet &amp; antibody of the invention. The label or package insert indicates that the composition is used to treat a particular condition. The label or package insert will additionally contain instructions for administering the antibody composition to the patient. Articles of manufacture and kits comprising the combination therapies described herein are also contemplated.

Package inserts are information that is typically included in commercial packaging of therapeutic products, containing information about indications, usage, dosage, dosing, contraindications, or precautions regarding the use of such therapeutic products. In other embodiments: the 'package insert indicates that the composition is for treating breast cancer, colorectal, lung cancer, renal cell carcinoma, glioma or nest cancer. The drug 2 may additionally comprise a second container, The container contains a buffer for medical acid sleep buffers, such as bacteriostatic water for injection (BWFI), phosphorus, and other materials required for commercial and user reasons... including other buffering swords, dilution swords, Over-the-counter also provides kits for a variety of purposes, such as for the injection of (four) 卩 or 142552.doc • J17- 201011047 HER2 from cell purification or immunosuppression. For separation and purification of vegf or HER2 'this kit can contain a VEGF/HER2 antibody (eg, 'bHl-44 or bHl-81) coupled to a bead (eg, agarose beads). It may be provided for inclusion in vitro (eg, by ELIS A or Western blot) and A kit for quantifying antibodies to VEGF or HER 2. As with articles of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container contains at least one of the multiple specificities of the invention. Antibody or A composition of a bulk fragment. May include an additional container containing, for example, a diluent and a buffer or control antibody. The label or package insert can provide a description of the composition and instructions for the intended in vitro or diagnostic use. The above written description is sufficient to enable those skilled in the art to practice the invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Various modifications of the invention will be apparent to those skilled in the art and are within the scope of the appended claims. Example 1. Library design and construction of antigen binding sites of antibodies by using three The heavy chain (HC) of the CDR loop recognized by the antigen is formed by the variable domain (Vh, association) of the light chain (LC). In many cases, one of the two variable domains (often Vh) is determined. Antigen specificity. Mice with a transgenic gene HC other than the intact Lc line produce neutralizing antibody valence (Senn et al., Eur. j. Claws... 33:950-961, 2GG3). Begin to investigate how bispecificity of antibodies can occur and whether different domains can achieve dual antigen binding specificity. 142552.doc .118· 201011047. Semi-empirical methods are used to find amino acid compositions for diversified antibody light chains. The design of the CDR length and library template, wherein the library template enables the production of a functional phage to display an antibody library and the selection of an antibody specific for binding to a protein antigen from the antibody library, as shown in the Kabat library. The sequence and length diversity of the CDR regions of the 1500 human-like kappa light chain sequence was used to guide the library design method. The residues exposed to the solvent were used for randomization. A subset of the randomized positions are customized to represent the amino acids at that site as part of the natural lineage, while the remaining sites are randomized to include all 20 naturally occurring amino acids. In particular, the light chain template (variable • domain) listed below (the underlined residues have been randomized) (SEQ ID NO: 10) is modified as described herein.

DIQMTOSPSSLSASVGDRVTITCIL^SOD^VNTAVAWYOOKPGKAPKLLIYS^ASFLYS

GVPSRPSGSGSGTDFTmSSLOPEDFATYYCOOH91YTTPPTFGOGTKVEIKRTVAAPS

VFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSFNRGEC

Four sets of libraries were generated based on 3 human Fab and scFv templates, with the dry heads in different groups for randomization (Figure 1). In all libraries, the heavy chain remains in the sequence determined by the library template

Constant. The heavy chain template (variable domain) sequence is listed below (SEQ ID NO: 11).

WQLVESGGGLVQPGGSLRLSCAASGFNIKDTYfflWVRQAPGKGLEWVARIYPTNG

YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW

GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALrS

GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

H-119- 142552.doc 201011047 Library design is summarized in Figures i and 2. All library templates contained a stop codon embedded in CDR L1 (sidhu et al., 2〇〇4) to prevent the presence of a template light chain between phage-presenting antibody library members. The template CDR sequence is outlined in circle 3. In one example, we introduce mutations in the lc variable domain of a HER2-specific antibody to identify variants that bind to different protein antigens while maintaining initial binding specificity. We adopt a conservative approach to randomize the LC CDRs to produce stable variants. Twelve solvent exposed LC CDR positions were selected for randomization: five of CDR1 (28, 29, 3〇, 31, 32), three of CDR2 (four of 50, 51, 53mcDR3 (91, 92, 93, 94). In addition, to guide the design of amino acid diversity at selected sites', the natural diversity of these positions was examined by analyzing approximately 1500 human-like κ LC CDR sequences (Johnson and Wu, Nucleic Acids ReS) 28:214,2000; Chothia and Lesk, J. Mol·Biol. 196:901, 1987) (Circle 4). Some locations with relatively high natural diversity (30, 3 1, 50, 92, 93) completely randomized, while other positions are limited to as few as two amino acid types to mimic natural antibodies. The length changes of native Lc CDR1 and CDR3 are also reflected in the library (Fig. 4) β is in Figure 4, χ indicates The type of amino acid designed at a low frequency is shown. Length diversity is constructed by inserting 1 to 5 residues between residues 3〇 and 31 and between residues 93 and 94. LC library is productive The original pedigree (Table 1). The listed are the results of 95 random pure lines at the end of the four-round selection. In detail, for the fixed standard Targets (VEGF, DR5, and human Fc) were selected for new binding specificity 142552.doc • 120- 201011047 (Sidhu et al., J. Mol. Biol. 338:299, 2004). After four rounds of selection, ELISA was used. The binding of 95 phage pure lines to the target HER2 and non-target protein (BSA) was assayed to ensure specific binding. The last round of selection of HER2 was performed for the enrichment of the target binding pure line that maintains HER2 binding. The positive pure line was sequenced. To identify the highest affinity conjugate, antigen binding IC5Q was determined by competitive ELIS A (Sidhu et al, J. Mol. Biol. 338:299, 2004). Shows the number of unique pure lines as determined by sequence analysis. And the number of unique pure lines (bispecific lines) that maintain HER2 binding. These lines display the lowest background binding signal for unrelated antigens (such as BSA). Table 1. Light chain library selection summary positive % Unique sequence HER2 positive human Fc fusion 63 31/61 1 hVEGF 77 41/74 30/41 DR5 length 85 5/82 2*/5 = weak binding signal target bispecific, screening for bispecific, selection of human Fc fusion 1/31 no hVEGF detected 30/41 94/94 DR5 Length 2*15 2/7** *=Weak Binding signal Her2 ** = weak binding signal DR5 is selected for the following three protein antigens: human vascular endothelial growth factor (hVEGF), death receptor 5 (DR5) and IgG complement-binding fragment (Fc), resulting in multiple binding lines ( Figure 5A). Some pure lines lose binding affinity for HER2, while other pure lines maintain HER2 binding and are therefore bispecific. Sequence analysis of 133 unique Herceptin® antibody variants with novel binding specificity recognizes amino acid substitutions and insertions compared to Herceptin® antibodies (Fig. 5B) 142 142552.doc -121- 201011047

The number of mutations is in the range of 3 to 17. The pure line (bispecifically pure line) that maintains HER2 binding contains on average fewer mutations than the pure line that loses HER2 binding ability. Maintaining the Herceptin® antibody CDR-L3 sequence is preferred but not sufficient to retain HER2 binding. This is consistent with the report that the Herceptin® antibody CDR-L3 is the most important LC CDR for HER2 binding (Kelley and O'Connell, Biochemistry 32: 6828 1993). Representative VEGF-binding pure lines were expressed as Fab and IgG proteins (Table 2). Table 2. Representative antibodies isolated from the light chain library of Herceptin® antibodies (SEQ ID NOS: 12-23). CDR-L1 CDR-L2 CDR-L3 specificity KD(nM) 2 8 2 9 3 0 3 0 a 3 0 b 3 0 c 3 0 d 3 1 3 2 3 3 5 0 5 1 5 2 5 3 9 1 9 2 9 3 9 3 a 9 3 b 9 4 Herceptin^ Γ) VN . - - TAVSASFHY ΐ '1' HER2 0.1 3-la NVWDWVPASSGWYIA VEGF 15 bHl Π TPRSISGYV w G s YHY '1' 1 VEGF/HER2 300/26 bH3 Π r GLG s V w A s YHYT '1' 19,000/8 bH4 DIRSG s V w G s YHYT r 3,500/11 3 The difference between the Herceptin® antibody and the Herceptin® antibody is shown in bold. To demonstrate that these antibodies specifically bind to their cognate antigens and do not interact non-specifically with other proteins, we demonstrate undetectable binding to a group of mammalian cell lysates and non-antigenic proteins. This assay confirmed the monospecific and bispecificity of purified IgG or Fab (Figure 6). The equilibrium binding affinity (KD) of a monospecific antibody derived from an LC library is in the range of 15 to 150 nM. The bispecific antibody binds to the new antigen (i.e., VEGF) with a high nM to low affinity and binds HER2 with low ηΜ affinity (Table 2). Among the antibodies shown in Table 2, antibody bH1 exhibited the highest bispecific affinity for two different protein antigens VEGF (KD = 300 nM) and HER2 (KD = 26 nM). Materials 142552.doc -122- 201011047 The enzyme and M13-K07 adjuvant sputum system was from New England Biolabs. E. coli XLl-Blue is from Stratagene. Bovine serum albumin (BSA), ovalbumin and Tween 20 lines were from Sigma. Neutral streptavidin, tropin and Superblock are from Pierce. The immobilized protein G and the anti-Μ13-conjugated horseradish peroxidase (HRP) line were from GE Healthcare (Piscataway, NJ). The Maxisorp immunoculture tray was from NUNC (Roskilde, Denmark). The tetramethylbenzidine (TMB) substrate is from Kirkegaard and Perry Laboratories (Gaithersburg, MD). All protein antigen lines were generated by a research group of Genentech, Inc. DNA degeneracy is indicated using the IUB code and, unless otherwise indicated, indicates a molar mixture: N=A/C/G/T, D=A/G/T, V=A/C/G, B=C /G/T, H=A/C/T, K=G/T, M=A/C, R=A/G, S=G/C, W=A/T, Y=C/T.

For example, at some randomized positions, the wild type is replaced by degenerate NNK codons encoding all 20 natural amino acids (N=A/T/G/C, K=G/T, etc. molar ratio). a. The XYZ codon refers to a codon having a unequal nucleotide ratio at each position of the codon triplet. X contains 38% G, 19% A, 26% T and 17% C; Y contains 31% G, 34% A, 17% T and 18% C; and Z contains 24% G and 76% C. Phagemid vectors for library construction Vector construction was performed using standard molecular biology techniques. Construct three templates for library generation. All templates are derivatives based on the plastid PV0354 used in the modified humanized 4D5 (type 8) heavy chain library (Lee et al., 2004a). By placing the 2C4 heavy chain variable domain to pV0354- containing the alkaline phosphatase promoter 142552.doc-123-201011047 (Lowman et al., 1991) and the stll secretion signal of both the light and heavy chains of Fab The 2C4 Fab-C template phagemid pJB0290 was constructed in a Fab-C vector. It was engineered to contain a monocysteine at the C-terminus of the heavy chain variable domain 1 to allow for the presentation of the bivalent Ml3 phage of 2C4 Fab as previously described (Lee et al., 2004b)» using Kunkel et al. The method (Kunkel et al., 1987) induced the introduction of the 2C4 light chain CDR into the Fab_e vector by site-directed mutagenesis. An epitope tag (gD tag) was added to the C-terminus of the light chain (Lasky and Dowbenko, 1984) to achieve a measure of the degree of presentation as described (Sidhu et al., 2004). The Fabl2-G library template PV1283 was generated by cloning the highly expressed heavy chain variable domain into pV0354-Fab-C, and the light chain variable domain was modified to contain Fab-12 (humanized A4.6.1, one CDR-L3 of anti-VEGF antibody). The highly expressed VH line is selected from the Fab library, which was transformed with Fab-12 (Y9〗STVPW96; SEQ ID NO: 24) using the CDR-L3 to induce alanine scanning mutation induction (Liang et al., 2006; Vajdos et al. Human, 2002), randomized heavy chain CDR residues of G6 Fab via panning against immobilized anti-gD antibodies. The design and construction of the divalent 4 (15(1^-foot 660)8〇?¥嗤嗤粒卩\^13 84 on the surface of the M13 phage particle was modified from the template pS201 8 described previously (Sidhu Et al., 2004) The seFv fragment contains a gD epitope tag in the linker region between the light and heavy bonds. The LC framework residue Arg66 is mutated to Gly66, which is in more than 95% of the native kappa light chain. Residues that are ubiquitous in this location, such as Kelley and

As described in Connell (Biochemistry 32: 6828, 1993), the mutation R66G only slightly reduced the binding affinity of the Herceptin® antibody to HER2 (&lt;2 fold). The CDR sequences of the library template are summarized in circle 3. 142552.doc -124· 201011047 Library Construction

Phage display libraries were generated using oligonucleotide directed mutagenesis as described (Sidhu et al., 2004). The library template vector contains a stop codon (TAA) embedded in CDR-L1, which is ligated to encode CDR-L1, CDR_L3 (all libraries), CDR-L2 (L1/L2/L3- during mutation-inducing reaction) Degenerate oligonucleotides on the sequences of A, -B, -C, +L4-D) and light chain framework 3 (L1/L4 and L1/L2/L3+L4-D) were repaired. The library mutation-inducing reaction was carried out according to the method of Kunkel et al. (Kunkel et al., 1987). The light chain CDR design for the library is shown in Figure 1, which summarizes the degenerate codons used at each position in the different libraries. For each CDR, three or four oligonucleotides were mixed at a specific ratio to encode the amino acid type of the desired frequency at each of the randomized and dry directions (Figure 4). The nucleotides are combined at different ratios to fine tune the diversity to reflect the amino acid frequency at the selected position in the native light chain kappa sequence. For CDR1, three oligonucleotides containing a codon at positions 91 to 94 were mixed at a ratio of 1:3:1: CAT NNK NNK RST (SEQ ID NO: 25) ' KMT XYZ XYZ RST (SEQ ID NO: 26) or DGG XYZ XYZ RST (SEQ ID NO: 27). XYZ is a variant of NNK that has an equal proportion of A/G/T/C at each site to reduce the coverage of aliphatic hydrophobic amino acids (Lee et al., J. Mol_ Biol. 340: 1073, 2004). For CDR2, four oligonucleotides containing a codon at positions 50 to 53 were mixed at a ratio of 1:1:2:10: NNK GST TCC NNK (SEQ ID NO: 28) ' TGG GST TCC NNK ( SEQ ID NO: 29), KGG GST TCC TMT (SEQ ID NO: 30) or NNK GST TCC TMT (SEQ ID NO: 31). For CDR3, each length is 142552.doc -125· 201011047

A mixture of 1:1:2 ratios of three nucleotides containing a codon at positions 28 to 33: G70A70C70 RTT NNK NNK TAC STA (SEQ ID

NO:32) ' G7〇A7〇C7〇 RTT NNK NNK DGG STA(SEQ ID

NO: 33) or G70A70C70 RTT NNK NNK NMT STA (SEQ ID NO: 34). G7〇A7〇C7〇 is a “soft” codon that allows 70% of the designated nucleotides and 10% of the other three nucleotides to encode about 50% of Glu and about 50% of other amino acids. Structural analysis of a large number of representative antibodies with κ LC revealed that CDR1 has the broadest range of conformation, which may be the result of changes in loop length (11 to 17 residues between positions 24 and 34). Different CDR-L1 lengths (length: 11 to 16) are therefore included in the library. The length of the native CDR-L3 also varies (length: 7 to 10 residues between positions 89 and 96), which is reflected by library design (length: 8 to 10; Figure 4). Figure 1 shows a comparison of natural diversity of light chains with actual library design. The mutation-inducing products of each library were combined in one reaction and electroporated into E. coli SS320 cells supplemented with K07 helper phage and grown overnight at 30 °C (Lee et al., J. Mol. Biol. 340) : 1073, 2004). About 1011 cells and about 5 to 10 pg of DNA were used in each electroporation reaction. The library phage was purified (Sidhu et al., J. Mol. Biol. 338:299, 2004). The number of transitions ranges from 1〇9 to l〇1G. The extent of presentation of intact Fab or scFv on the surface of the phage was determined by ELISA binding assay, in which 96 of the purely lined anti-gD antibodies were randomly selected from each library. The degree of presentation is in the range of 5% to 25% (circle 2). The pure 25°/◦ of the antibody is present to maintain HER2 binding. The sequence of 142552.doc -126-201011047 was determined for approximately 150 rendered lines to verify actual library diversity compared to design diversity. Due to incomplete mutation induction (the template stop codon in CDR-1 ensures 100% mutation of this CDR in the scFv expressed), a portion (about 30%) of the functionally presented library members retain the Herceptin® antibody CDR-L2 and / Or CDR-L3 sequence. These were excluded from the sequence analysis of actual library diversity. At most of the randomized positions, the diversity of the phage display pools presenting pure lines did not significantly deviate from the design diversity (ρ &gt; 0.05, odds rati〇 test). The exception is position 29 of CDR-L1 (where vai is found to be slightly overextended (p=〇_〇〇5) compared to ne) and positions 51 and 53 of CDR-L2 (where Gly and Ser are respectively compared to Ala and Tyr is more common (p &lt; 0.01)). Example 2. Evaluating library potency Library sorting and screening When an antibody that specifically binds to various protein antigens can be isolated after 4 to 5 rounds of selection, a library is considered functional. A variety of protein targets are known to allow for functional immobilization for library panning and specific antibodies have been generated from validated phage display libraries (Fellouse et al, 2005) (Lee et al, 2004a). To evaluate each set of libraries, we selected a subset of these targets for selection (Circle 2). The libraries are subjected to a first round of binding selection wherein the anti-antibody or protein L serves as a capture target to remove the pure line of the deleted Fab/scFv gene, followed by 4 to 5 rounds of antigen selection. Alternatively, it is directly subjected to target binding selection without pre-selection of anti-gD or protein L. NUNC 96-well Maxisorp plates were coated with antigen (5 pg/ml) overnight and blocked with alternating blockers for 1 hour (Figure 7). In the first selection cycle, ι η phage/ml of the phage solution was added to the coated immunoculture dish. 142552.doc -127· 201011047 Reduce phage concentration in each round of selection. After incubating the phage solution on the immunoculture plate to allow binding to the immobilized antigen, the plate was washed repeatedly with PBS, 0.5% Tween 20. In order to increase the stringency, reduce the incubation time in each round of selection (4 hours in the first round, 3 hours in the second round, 3 hours in the third round, 2 hours in the 4th round, 1_75 hours in the 5th round) and increase the number of washings (Figure 7). The bound phages were lysed with 0.1 M HCl for 30 minutes and the lysate was neutralized with 1.0 M Tris base. The phage recovery of each antigen-coated immunoculture well was calculated and compared to the phage recovery of the uncoated antigen-blocked wells to study the presence of Fab or scFv cells that specifically bind to the standard antigen. Pure enrichment (circle 7). The lysed phage were expanded in E. coli and used for other rounds of selection. Random pure lines from rounds 4 and 5 were selected for screening and assayed using phage ELISA, where binding to the target and anti-gD was compared to binding of non-related protein (BSA) to test for non-specific binding. . A pure line that binds to an anti-gD antibody and a target but does not bind to a non-specific protein is considered to be specifically positive. The libraries L1/L3, L1/L4, L1/L2/L3-A, Ll/L2/L3-B_l and Ll/L2/L3-B_2 do not produce any specific positive pure lines, while the library L1/L2/L3-C and L1/L2/L3+L4-D makes it possible to isolate antibodies specific for the target antigen. For example, a fourth round of random lines was assayed using phage ELISA, in which the binding specificity was tested by comparing the binding of the individual amplified lines to the binding of the target and HER2 to the binding of the non-target protein (BSA). For enrichment of the HER2-binding phage line, the lysed phage from the third and fourth rounds of VEGF or DR5 selection were amplified and subjected to another round of selection on HER2 coated wells. The VL and VH regions of the positive pure lines were amplified by PCR and subjected to the 142552.doc-128-201011047 sequence. The hit rates of hFC, hVEGF and hDR5-lf were 63%, 77% and 85%, respectively. The positively pure VL region was amplified by PCR and sequenced as described (Sidhu et al., 2004). DNA sequence analysis of positive specific binders revealed that the percentage of unique pure lines was 51% (hFC), 55% (hVEGF) and 6.1% (hDR5-lf). The sequence of the unique hVEGF binding line is summarized in Figure 8. Combination of hVEGF and pure lines and solution selection

A high diversity of hVEGF-binding pure lines was observed following four rounds of sorting. To identify high-affinity hVEGF-binding pure lines, a solution-based selection method was employed after the fourth plate-based sorting. 50 nM biotinylated hVEGF was incubated with phage selected for propagation from the fourth round of immobilized antigen. After incubation for 2 hours at room temperature under shaking, the hVEGF-binding phage were captured on a neutral streptavidin-coated and blocked immunoculture dish, followed by repeated washing. The phage is purely lysed, screened and sequenced as previously described. The sequence of the hVEGF-binding pure line from the final solution selection step is shown in Figure 9. Separate bispecific pure lines from library L1/L2/L3-C and L1/L2/L3+L4-D for library L1/L2/L3-C and L1/L2/L3+L4-D library template from hu4D5 The scFv fragment was modified from the antibody, which binds Her2 with high affinity. The functional complement of Her2 binding for hu4D5-5 was induced by alanine scanning mutation in the CDR regions, which showed that the heavy chain residues supply most of the binding free energy, while the supply of individual light chain residues is Smaller (Kelley and O'Connell, 1993). Analysis of the atomic structure of the Herceptin® antibody Fab complexed with human Her2-ECD showed that although the light chain involved 142552.doc-129-201011047 and produced antigenic contact, the heavy chain provided most of the structural interface with the antigen (Cho et al. , Nature 421:756, 2003). We observed that some members of the functional light chain library constructed on the Herceptin® antibody template retain Her2 binding ability. Two strategies were used when attempting to separate the bispecific scFv fragment capable of binding Her2 and the second antigen from the functional libraries L1/L2/L3-C and L1/L2/L3+L4-D. In one method, a positive pure line that retains Her2 binding is screened by ELIS A from a positive pure line selected from the target antigens previously described. The percentage of specific positive lines capable of binding to Her2 varies depending on the specificity of the second antigen. Only 1/61 of the unique hFc-specific positive line still binds to Her2 (1.6%), 30M1's unique hVEGF-binding line still binds to Her2 (73%), and 2/5 of the unique hDR5 conjugate still binds Her2 (40°/〇) ). In addition, a bispecific antibody was isolated by selection of a Her2 conjugate from a pool of hVEGF and hDR5 binding antibodies using a selection-based approach. The isolate from the 4th round of target antigen sorting was subjected to another round of selection by incubating 2xl013 phage/ml for 1 hour on a Heriso coated immunoculture plate coated with Her2 (5 pg/ml). The plate was washed 15 times with PBS, 0_5% Tween 20, and the bound phages were dissolved as described above. Random random lines were selected and Her2, anti-gD, and target binding were assayed and compared to non-specific binding of non-related proteins (BSA). All 192 tested pure lines were confirmed to be specific positive and sequenced as previously described. Sequence reveals 94 unique sequences. In summary, the method produced 94 Her2/hVEGF bispecific lines (100%) in 94 unique lines tested (Figure 8). The sequences of all isolated unique hVEGF/Her2 bispecific antibodies from both isolation strategies are summarized in Figures 10A and 10B. 142552.doc -130- 201011047 The sequence of the isolated pure line that lost all detectable binding to Her2 is shown in circle 11. In the pure line with dual specificity, almost all of the pure lines retain the Herceptin® antibody CDR-L3, which makes it possible to maintain CDR-L3 essential for maintaining HER2 binding. In the case of hDR5, 2/7 of the unique Her2 binding pure line was bispecific (29%, 12 sequenced lines). One of the dual specific lines of CDR-L3 has some homologous changes. High yield characterization of hVEGF binding pure line

A high yield single point competitive ELISA (Sidhu et al., 2004) in a 96-well format was used to screen for pure lines with high affinity for hVEGF and to study the VEGFR1 blockade profile. Briefly, Maxisorp immunoculture plates were coated overnight at 4 °C with 2 gg/ml hVEGFi〇9 and blocked with 1% (w/v) BS A for 1 hour. The phagemids in E. coli XLl-Blue were grown in 150 μL in 2 ΥΤ medium supplemented with carbenicillin and Μ13-Κ07 helper phage; the culture was shaken at 37 °C at 96 °C. The pore format grows overnight. The phage-containing culture supernatant was diluted five-fold in PBST (PBS with 0.05% Tween 20 and 0.5% (w/v) BSA) with or without the addition of 100 nM hVEGF109 for affinity screening. For receptor block screening, hVEGF-coated wells were incubated in the presence or absence of VEGFR1 domain l-3 (Dl-3) and VEGFR1 domain 2 (D2), and then added on a 5-fold diluted phage Clear liquid (Liang et al., 2006; Wiesmann et al., 1997). After incubation for 1 hour at room temperature (RT), the mixture was transferred to a hVEGF1()9 coated plate and incubated for 10 minutes. The plates were washed with PBT (PBS with 0.05% Tween 20) and incubated for 30 minutes with 5,000-fold diluted anti-Ml3 antibody horseradish peroxidase conjugate in PBST for 142552.doc-131-201011047. The plates were washed, developed with TMB for approximately five minutes, quenched at 1.0 Μ Η 3 Ρ〇 4, and read spectrophotometrically at 450 nm. In the single-point affinity assay, the ratio of the absorbance in the presence of the solution phase hVEGF! 〇9 to the absorbance in the absence of the solution phase hVEGF1()9 is used as an indicator of affinity. A low ratio will indicate the majority of the Fab phage. It binds to the solution phase hVEGF109 at the initial incubation stage and therefore cannot be captured by immobilized hVEGF1()9. The results of the first 41 unique pure line high yield affinity tests are summarized in circle 12. Similarly, for the blocking assay, a low ratio indicates that the binding of the pure line to hVEGF109 is blocked by the hVEGF109-VEGFR1 interaction, indicating that some of the pure lines have overlapping binding sites on the VEGF with the respective VEGF receptor fragments ( Antigenic determinants) (Figures 13A and 13B), and such pure lines may exhibit blocking antibodies. High Yield Characterization of Bispecific h VEGF/Her2 Pure Lines The same principles as described in the previous section were used to achieve isolation of pure lines with high affinity for hVEGF and Her2 for further characterization (Fig. 14A). High-yield single-point competitive ELISA was used to coat Maxisorp immunoculture plates overnight at 4 °C with 2 pg/ml hVEGF丨〇9 and Her2-ECD, followed by 1% (w/v) BSA for 1 hour. To isolate the pure lines with high affinity for hVEGF and Her2. Phage-only lines that were identified as bispecific in previous single-point ELISA screening were grown as described previously and were incubated with and without the addition of 20 nM Her2-ECD and 50 nM hVEGF. After incubation for 1 hour at room temperature, the solution was applied to the coated immunoculture dish and the binding signal was recorded and analyzed as described in the previous section. A pure line with low ratios for both hVEGF and Her2 was selected for further characterization. The lowest signal ratio hVEGF-specific and hVEGF/Her2 bispecific phage pure lines and the DR5 binding and DR5/Her2 bispecific from the initial single-point ELISA screening were selected in the single 142552.doc -132- 201011047 point competitive ELISA. Phage-pure lines and VEGF-binding pure lines from combinatorial plates and solution selection were used for affinity measurements by competitive ELISA. Phage pure lines were propagated from a single colony by growing overnight at 25 °C in 2 ml of 2YT culture supplemented with capnicillin and K07 helper phage. The phage purified by precipitation in PEG/NaCl was first serially diluted in PBST and tested for binding to the antigen coated culture plate. A dilution that produces a 50% to 70% saturation signal is used in the solution binding assay, wherein the phage is first incubated with the increased concentration of antigen for one to two hours and then transferred to the antigen coated plate '10 to 15 Minutes to capture unbound phage. IC5Q is in solution

The binding phase inhibits the concentration of antigen bound to the immobilized antigen by 50% of the phage (Lee et al., 2004a). Figure 14B shows a curve that can be used to calculate the analyzed hVEGF-binding pure IC5(R) from the culture dish sorting strategy. The IC50 value is in the range of 22 nM to more than 1 μΜ (Fig. 14B). The IC50 values for the hVEGF conjugates isolated using the combination plates and solution based selection ranged from 41 nM to 226 nM (Figure 9). The IC50 value of the DR5 binding pure line is in the range of 20 nM to more than 1 μΜ. The IC50 values for the hVEGF/Her2 bispecific strain are summarized in Figure 15. Example 3. Characterization of antibodies from the light chain library Conversion of scFvs to Fabs To test whether the conversion of scFvs'2 to Fabs presented on phage affects the affinity of the binding line in the library, two pure lines (3-7 anti-hVEGF and 4-1 142552.doc -133- 201011047 Anti-hDR5) was used to convert to Fab and present on bacillus. The VL region of the sputum granule DNA of the selected hVEGF and DR5 scFv fragments is digested with restriction enzymes, and the enzyme cleaves the upstream of the DNA encoding the region of CDR-L1 (five and downstream of the region encoding CDR_L3 (foot p«I). The digested DNA fragment is ligated to a similarly digested vector (PAP2009) that has not been used for Fab hu4D5 by the fusion of the C-terminal domain of the M13 gene _3 minor outer sheath protein (Lee et al. 2004b). The resulting bicistronic sputum granule contains a light chain fused to the C-terminus and the epitope (gD) tag and a heavy chain (VH and CH1) in which the C-terminus and the Ml 3 minor outer sheath protein (p3) are fused. Under the control of the auxin citrate promoter, the first open reading frame encodes a secretion signal from stI1 followed by a Fab4D5 light chain (wherein the CDRs are replaced by 3-7 anti-hVEGF and 4-1 anti-hDR5 scFv'2 CDRs) The polypeptide consisting of a gD-tag epitope, the second open reading frame encoding a fusion polypeptide consisting of the stH secretion signal, the Fab4D5 heavy chain, the amber (TAG) stop codon, the Gly/Ser linker sequence, and the g3 protein C-terminal domain (cP3). In E. coli XL-1 Blue co-infected by M13-K07 The performance resulted in the production of Ml3 phage displaying Fab variants of 3-7 and 4-1 scFv'2. Competitive phage ELIS As was used to estimate IC50 values of phage-presented scFvs and Fabs affinity for hVEGF and hDR5. The format data is quite consistent (data not shown). To present bHl Fab on the surface of M13 phage, the plastid pAP 2009 was modified to encode bHIFab. The variant of bHl Fab was used as a library template, which was respectively in the LC and HC pools. Stop SEQ ID NO: 142552.doc •134- 201011047 2002). The degeneracy ranged from 1χ105 to 1χ108, and the actual library size ranged from 6xl09 to 4χ101(). Each library was constructed as described above. Targeted for immobilized targets (VEGF, HER2-ECD, protein L) Or anti-gD mlgG) for two to three rounds of selection (Vajdos et al, J. Mol. Biol. 320: 415, 2002). For target binding, phage ELIS A screening targets bind to the pure line, followed by DNA sequencing And sequence alignment to calculate wild type/mutation at each position Ratio. For presentation and protein folding effects, the ratio of sequence analysis of approximately 100 unique sequences of VEGF and HER2 binding to the pure line is corrected by dividing the ratio calculated by the sequence of more than 100 anti-gD binding lines to obtain F wild type 4 / Burst * value. When only the Fab heavy chain is fused to the phage coat, the phage exhibits a gD tag fused to the light chain indicating proper folding and association of the light and heavy chains. Consistent with this • The binding of protein L to the nonlinear epitope on the Fab light chain also results in a wild-type/mutant ratio similar to the gD-tag selection. AAG=RT/« (Ka, wild type/Ka, mutant si jsRT/wfF wild type/mutant wild type/mutation) as described in Vajdos et al. (J. Mol. Biol. 320:415, 2002) type

The value is converted to AAG.

Characterizing library conjugates as free human Fab and IgG To accurately determine the affinity, specificity, and other properties of antibodies, select representative pure lines of each specificity group that exhibit the highest affinity in a competition ELISA assay for performance Free Fab and hlgG (Figure 16). The variable domains of the light and heavy chains are housed in vectors previously designed for Fab expression in E. coli or transient human IgG expression in mammalian cells (Lee et al, 2004a). The Fab protein was produced by growing the transformed 34B8 E. coli cells in intact C.R.A.P. medium at 142552.doc -135-201011047 for 26 hours as described (Presta et al., 1997). hlgG was expressed by transient transfection of 293 cells, and hlgG was purified by protein A affinity chromatography (Fuh et al, J. Biol. Chem. 273: 11197, 1998). 1 L of E. coli culture was purified by protein G affinity stratification. The column was washed with PBS and the Fab protein was lysed with 1 mM NaOH and dialyzed against PBS. 4 L E. coli cultures were subsequently purified by cation exchange chromatography on Protein A affinity columns as previously described (Muller et al., 1998). The protein concentration was determined spectrophotometrically. The final yield of F ab obtained from the purification of small scale shock flask flasks is generally from 0.8 to 15 mg/1. The IgG yield in the small-scale culture was a medium to high yield of 6.7 to 60 mg/1 (Fig. 17). The purified protein was first characterized using size exclusion chromatography and light scattering to ensure that the protein did not exhibit significant levels of protein aggregation (&lt; 5%). Briefly, Fab and hlgG expressed by binding to the respective antigens were screened by ELISA. All variants except one variant were found to bind to their cognate antigen. Pure line 4-6 loses hDR5 binding ability when converted to Fab and hlgG. Binding of selected anti-VEGF pure lines to hVEGF165 produced against shorter forms of hVEGF109 using standard ELISA (H3, H4_N, H4_D hlgG) and competitive ELISA (bH1, 3-1, 3-6, 3-7 hlgG) . G6 hIgG (Fuh et al., 2006) was used as a positive control (Figs. 18A and 18B). All pure lines bind to hVEGF165 as expected. To study the degree of protein aggregation, as with purified Fab and IgG, the selected pure lines were analyzed by size exclusion chromatography (SEC) followed by light scattering (LS) analysis. Samples at a concentration of 0.5 mg/ml (hIgG) and 1 mg/ml (Fab) were assayed in PBS. For all samples, a maximum of 142552.doc • 136· 201011047 5% aggregation was observed at a given concentration (Figure 17), which was within the range previously observed for other phage-derived antibodies. Pure lines 3-6 and 3-7 did not appear at the expected time points, indicating that the reformatted 1 gG and Fab exhibited aggregation and/or non-specific interaction with the resin (data not shown). These pure lines were removed from the pure collection that was subjected to further analysis.

To exclude cross-reactive and non-specific binding, we used a standard ELISA assay to study high concentrations (1〇〇nM) of selected hlgG with a set of immobilized protein targets including whole cell lysates, homologous antigens and homologs. Binding of the target. In addition to the antigen, we immobilized murine variants of hVEGF to test the anti-hVEGF pure line and species reactivity. In detail, the histones were immobilized on Maxisorp plates and blocked with 1% BSA in PBS for 1 hour. The hlgG (or Fab) was diluted to a concentration of 100 or 500 nM in PBST and transferred to a coated plate. The plate was washed after 1 hour of incubation and cultured with protein A that binds HRP. The binding signal was developed by addition of TMB receptor for approximately 5 minutes, quenched with 1 M H3P〇4 and read spectrophotometrically at A45. The tested hlgG specifically binds to its antigen. The pure lines bHl and 3-1 showed reciprocal reactivity with murine VEGF (mVEGF) (Fig. 19). To test whether the bispecific antibodies bH1, H3 (anti-hVEGF/Her2) and D1 (anti-hDR5/Her2) can simultaneously bind to their cognate antigens or whether the antigens compete for antibody binding, hVEGF is fixed at a concentration of 2 pg/ml and hDR5. A fixed concentration of hlgG was incubated with serial dilutions of Her2-ECD, which in turn captured hlgG on the immobilized antigen. In each case, Her2-ECD binding was found to compete with binding to other antigens (Figure 20). To accurately determine the affinity of IgG and Fab (ie, anti-hVEGF isolated from the library and 142552.doc-137-201011047 anti-hVEGF/Her2 Fab and IgG) and to study the immediate binding profile, we used BIAcoreTM-3000 (BIAcore, Uppsala, Sweden) Surface plasmon resonance (SPR) assays were performed on immobilized hVEGF, mVEGF, DR5 and Her2-ECD CM5 sensing wafers on a machine with 40 to 300 reaction units (RU) depending on the analytes studied. Immobilization was carried out as described (Chen et al., 1999). To minimize the affinity effect of the bivalent IgG analyte, in which case the lower density ligand is targeted to the sensing wafer" injection at a rate of 22 to 30 μΐ/min is approximately below the estimated KD ( The binding reaction was corrected by subtracting the RU of the reference flow cell by subtracting the RU of the reference flow cell based on a 10-fold increase from the competition ELISA experiment to an increasing concentration in the 10-fold concentration range of the estimated KD. In addition, the reaction was double referenced to correct instrument bias by subtracting the RU of the flow cytometry of the injected ligand with the sample buffer (PBS with 0.05% Tween 20). For the kinetic analysis of Fab, a 1:1 Langmuir binding model was used to calculate &amp; 011 and A; 〇ff. When necessary (high analyte concentration), a 1:1 Langmuir binding model with mass transfer limitations was applied. A bivalent analyte binding model with or without mass transfer restriction was used for IgG analytes (BIAcore Evaluation Software 3 2). In the case of H3 hlgG, H4_N Fab and H4_DhIgG, the fit of the reaction to the kinetic binding model was unsatisfactory. Therefore, a steady state binding assay is applied in which the equilibrium response is plotted against analyte concentration. Estimate KD based on ECso. A summary of the BIAcore binding analysis can be found in Figure 21. The affinity of hVEGF-binding antibodies 3-1, 3-6 and 3-7 was found to be in the range of Nemo. The bispecific antibodies (bHl, H3, H4-N, H4-D) analyzed showed low micro-Mor to micro-Mole affinity for hVEGF. In contrast, the affinity for Her2 is in the range of 8 to 142552.doc -138.201011047 59 nM (Fab). To determine whether the anti-hVEGF conjugates bH1, H3, and Η4_Ν2 light chains bind to hVEGF, regardless of the sequence of the relevant heavy chain, the light chain is variable by cloning the light chain variable domain into the 2C4 Fab expression vector pJB0524 The domain was grafted to the anti-Her2 2C4 Fab, thereby replacing the 2C4 light chain variable domain. Fab is expressed as described above. The bHl/2C4 and H3/2C4 chimeric Fabs were not expressed in detectable amounts. The H4_N/2C4 chimeric Fab protein was isolated and tested for binding to hVEGF (bHl initial specificity) and Her2 (bHl, 2C4 initial specificity). No binding to hVEGF and Her2 was detected by standard ELISA binding assay (Figure 22). The results indicate that the heavy chain of bHl is required for antigen binding. Comparison of anti-hVEGF epitopes

To roughly locate the epitopes of anti-hVEGF antibodies on hVEGF, we investigated the ability of these newly isolated anti-VEGF antibodies to compete with other hVEGF-binding antibodies and compete with VEGF receptors for known binding sites (Fuh et al., 2006). Muller et al., 1998; Wiesmann et al., 1997). The assay was performed in a competitive £1&gt;18 format, in which the VEGFR1 (Flt) domain 1-3 and the anti-hVEGF antibody were immobilized on the immunoculture plate by 2 §/1111 on the ^3乂丨3〇 print.

Avastin® (IgG), B20-4.1 (IgG), G6 (Fab) and KDR domain 1-7 Fc fusion proteins. The solution competition binding assay uses biotin-labeled VEGF equilibrated with serial dilutions of purified IgG protein and captures unbound biotin-VEGF with immobilized Fab or IgG coated on a Maxisorb plate and binds to the anti-protein chain HRP detection of bacteriocin (Lee et al, J. Mol. Biol. 340: 1073, 2004). Antibodies that block the binding of hVEGF to other hVEGF binding antibodies or hVEGF receptors may share overlapping epitopes. High concentration of 142552.doc -139- 201011047 (μΜ) bispecific hVEGF/Her2 binding antibody (bH1) can completely block the binding of hVEGF to its receptor (VEGFR1 and VEGFR2), indicating bH1 epitope and VEGFR1 ( Circle 23) and VEGFR2 (circle 23) overlap sufficiently. In addition, bH1 blocked hVEGF binding to B20-4.1 (Fig. 24). H3, H4_N &amp; H4_D also blocked hVEGF binding to both receptors, indicating that its antigenic determinant is similar to bHl (circle 23). An incomplete blockade profile may be the result of its relatively low affinity for hVEGF (Figure 21). In contrast, 3-1 did not block the binding of hVEGF to VEGFR1 even at the highest concentration (0.5 μΜ) (Figure 23). In addition, we were unable to detect 3-1 hlgG #11 blockade of Avastin® antibody (Figure 25). However, 3-1 hlgG blocks the binding of hVEGF to VEGFR2 (KDR) (circle 23) and Β20-4·1 (circle 24). These results indicate that 3-1 has a unique epitope compared to other antibodies. Example 4. Structure-function study of bHl, anti-hVEGF/Her2 bispecific antibodies

Structural and functional studies were performed to explain the nature of bHl's interaction with its two antigens (VEGF and HER2). The Herceptin® antibody differs from bH1 in CDR-L1 (V29NTA32 vs. I29PRSISGY32; SEQ ID NOS: 35 and 36) and CDR-L2 (S50ASF53 vs. W50GSY53; SEQ ID NOS: 37 and 38). bHl anti-VEGF/Her2 was selected for structural characterization based on its dual specificity and its relatively high affinity for VEGF and Her2. To study the functional and structural epitopes on VEGF and Her2, we have crystallized bHl Fab complexed with the VEGF1()9 and !iHer2 extracellular domains and resolved the structure of the two complexes by X-ray crystallography. In addition, 'we used a combination of alginoside and homologous shotgun scanning analysis as described in (Vajdos et al. 2002). 142552.doc -140- 201011047 bHl Fab Expression, Purification, Crystallization, and Data Collection Receptor binding moieties consisting of residues 8-109 that are expressed, refolded, and purified as described previously (Christinger et al., 1996). Residues 1-624 of the extracellular domain of Her2 were expressed and purified as previously described (Franklin et al., 2004; Hudziak and Ullrich, 1991).

Whole cell pellets were obtained from 10 liters of E. coli fermentation for large scale bHl Fab preparation. 220 grams of cell paste was thawed into 1 L PBS, 25 mM EDTA, 1 mM PMSF. The mixture was homogenized and then passed through a microfluidizer. The suspension was then centrifuged at 125 ml for 90 minutes in a 250 ml aliquot. The protein was then loaded onto a Protein G column (25 ml) equilibrated with PBS at a rate of 5 ml/min. The column was washed with equilibration buffer and then dissolved in 0.58% acetic acid. The fraction was determined by SDS • PAGE (data not shown). The fraction containing bH1 Fab was fractionated and then loaded onto a 5 〇 ml cation exchange SP saccharide column (Pharmacia) equilibrated at 20 mM ME S (pH 5.5). The Fab was dissolved in a gradient of sodium vapor in the equilibration buffer. The gradient was linear to 0.5 M NaCl, 20 mM MES (pH 5.5). Fractions containing Fab were identified by SDS-PAGE (data not shown) and combined. The bHl Fab was eluted at a NaCl concentration of about 0.5 Torr. The Fab concentration was determined by measuring A28g. The final yield of bHl Fab was 67 mg/1 酦 yeast growth. The complex was obtained by mixing the purified bHl Fab with VEGF or Her2 ECD at a molar ratio of 2:1, and for the VEGF-Fab complex at 25 mM D 1^-11 (:1 (? only 7.5) And 0.3] sodium chloride was purified by size exclusion chromatography (SP-200, Pharmacia) and for Her2 ECD-Fab complex 142552.doc • 141 - 201011047 with 25 mM Tris-HCl (pH 8 And 0.15 Μ gasification sodium purification. The composition of the obtained complex was examined by SDS PAGE (data not shown). The protein complex was concentrated and used in the crystallization test. In the case of bHl-VEGF complex, at 19° The initial hanging drop experiment using the vapor diffusion method under C produced small isomorphous crystals from 14 different conditions in one week. The crystals of the bHl-Her2 complex appeared in four conditions within one week. In each case, the selection was from one condition. The crystals were used for further optimization. For the crystal of bHl Fab-VEGF (8-109), an equal volume of protein complex solution (10.6 mg/ml protein, 300 mM NaCl, 25 mMTris-HCl (pH 7.5) )) mixed with crystallization buffer containing 0.15MD, L-malic acid (pH7.0), 20% PEG335(R), and equilibrated at 19 °C After 24 hours, a large crystal belonging to the space group C222! having a unit cell size of a = 100.6, b = l98.0, and c = 77.7 appears. The crystal form contains 1 Fab and 1 VEGF single in the asymmetric unit. Before the data was collected, the crystals were frozen by liquid droplets in the artificial mother liquor containing 5%, 10% and 15% glycerol, followed by rapid freezing in liquid nitrogen. Advanced Light Source (Berkeley) beam line 5·0·1 collects data for 2.6 A. By using a protein solution (11 mg/ml, 25 mM Tris (pH 8) and 150!111^ sodium) with 25°/( The crystals of bHl Fab-Her2 (l-624) were obtained by mixing crystallization buffers of 0.11^14£8 (卩116.5). After 12 hours, it appeared to have a=62.3, b =115.1, c=208.2 unit cell size space group P2JP! crystal. The crystal contains a Her2-Fab complex in the asymmetric unit. Before data collection, the crystal is 20% ethylene glycol 142552.doc -142 - 201011047 Rapid freezing in liquid nitrogen as a cryoprotectant. Data was collected for 2.9 A using the beam source 5.0.1 of the Advanced Light Source (Berkeley). Data processing, structure determination and improvement Data were processed using Denzo and Scalepack (Otwinowski, 1997). The structure of the bHl Fab complex was analyzed by Phaser (L. C. Storoni, 2004; Read, 2001). Resolution of VEGF from the previously described VEGF-Fab complex (2FJG) and Fab fragment containing the variable domain VL/VH or constant domain Chi/Cl of the Herceptin® antibody Fab-Her2 complex (1N8Z)

bHl-Fab-VEGF (8-109) complex. When the bHl-Her2 structure was analyzed, the fragment of Her2 and the variable domain of the Herceptin® antibody Fab from the Her2-Fab complex 1N8Z were used as a search model. The constant region of the bHl Fab could not be found using the Herceptin® antibody Fab constant portion as a search model (1N8Z) and must be manually docked by the Herceptin® antibody Fab-Her2 complex structure. Model creation and improvement were performed using the programs Refmac (Collaborative Computational Project, 1994) and Coot (Emsley and Cowtan, 2004). Stereochemical parameters were analyzed using MolProbity (Lovell et al., Proteins 50: 437 (2003)). The structure was modified for the Fab-VEGF complex to IU = 0.22 and R* * = 0.27, and the structure was modified for the Fab-Her2 complex to have an R value = 0.25 and R® * = 0.31. The crystal structure of bHl Fab complexed with VEGF and Her2-ECD was modeled. Some bHl Fab residues are within 4.5, 4.0 and 3.5 A of the antigen. The two paratopes of the two antigens on the same antibody (the region of the antibody that is in contact with the antigen) overlap significantly and the residues from both the light and heavy chains are involved in binding to the two antigens. bHl binds to an epitope on VEGF that is similar to the Avastin® antibody, and bHl binds to 142552.doc •143- 201011047

Herceptin® antibodies are essentially identical to Her2 on the epitope. The crystal structure of bHl Fab bound to HER2 extracellular domain (ECD) (residue 1-624) and VEGF receptor binding domain (residue 8-109) was determined by resolution of 2.9 A and 2.6 A, respectively (Fig. 26 and Table) 3). Figure 26 shows the crystal structure of the bHl Fab/HER2 crystal structure and the bHl Fab/VEGF complex superimposed with the Herceptin® antibody/HER2 complex. Table 3. Crystallographic studies bffl Fab/hVEGF complex bHl Fab/HER2-ECD complex data collection statistical space group C222, P2! 2i2i unit cell (A) a=100.6, b=198.0, c=77.7 a=62.3 , b=115.1, c=208.2 Beamline, wavelength ALS 5.0.1 ALS 5.0.1 Resolution (person) 50.0-2.6 50.0-2.9 Rsyma 0.090 (0.66) 0.095 (0.66) Number of observations 151680 192951 Unique reflection 24705 34149 Integrity (%)* 99.8 (100) 100(100) 1/σ(1)* 16.0(3.0) 18.5 (2.6) Improving the content of the statistical asymmetric unit 1/2 VEGF dimer, 1 Fab 1 Her2-ECD single Body, 1 Fab resolution (human) 30.0-2.6 30.0-2.9 Reflection 22977 32277 R factor b, R free 0.19, 0.25 0.22, 0.28 RMS deviation bond (person) 0.011 0.010 RMS deviation angle (°) 1.3 1.3 Ramachandran statistically favorable Zone (%) 96.5% 89.9% Allowable zone (%) 99.4% 97.9% Outliers (%) 0.6% 2.1% Number of residues 528 1017 Number of water 49 0 Number of sugars 0 2 Number of ligands/ions 1 Glycerin) 1 (MES)

Rsyma=EI-&lt;I&gt;ΣΙ.&lt;Ι&gt; is the average intensity of symmetrically related observations of unique reflections. R® Jb= Σ F°-Fc ΣΙ F0. The R-free system indicates the value of the highest resolution shell based on values in brackets other than the 5% reflection excluded from all improvements. 142552.doc -144- 201011047 In the bHl/HER2 complex, the Fab binds to the domain of HER2 in a manner similar to the Herceptin® antibody (Cho et al., Nature 421:756, 2003); the two complexes are 2.3 The root mean square deviation (rmsd) of the Ca position of A is superimposed. In VEGF complexes, bH1 recognizes epitopes that overlap with the binding sites of VEGF receptors (VEGFR1 and VEGFR2) and other VEGF antibody binding sites (Wiesmann et al, Cell 91:695, 1997; Muller et al, Proc. Natl. Acad. Sci. USA 94:7192, 1997). Consistent with this, it was observed that bH1 blocks the binding of VEGF to its receptor (Fig. 27). For the data shown in Figure 27, biotinylated human VEGF165 was equilibrated with increasing concentrations of IgG (x-axis). Unbound hVEGF165 was captured on the immobilized VEGFR2-ECD Fc fusion protein and detected spectrophotometrically (optical density at 450 nm, y-axis).

As shown in Figure 28, the binding sites of VEGF and HER2 on bH1 were broadly overlapped. Twelve of the four residues that bite HER2 also contact VEGF. The two binding sites include CDR residues from HC and LC. In the HER2 complex, the LC and HC CDRs provided approximately equal antigen contact regions (53% and 47%, respectively), whereas in the VEGF complex, the LC CDRs constituted nearly 70% of the embedded surface (Figure 29). The Herceptin® antibody and the HER2 binding site on bHl are similar, and differ only in the CDR-L1 and -L2 regions, where the Herceptin® antibody sequence is not conserved in bH1 (Figure 28). In Figure 28, 'blocking the residue on the surface of bHl or Herceptin® antibody Fab according to the degree of embedding by VEGF or HER2 (dark shade and white text &gt; 75% embedding, moderate shading and white text 50-75%) Embedded, light shades and black text 25-49% embedded). The underlined residues are not between bHl and Herceptin® antibodies. 142552.doc •145· 201011047 The same. The white dashed line shows the separation of the light chain from the heavy chain. The configuration of the bHl Fab complexed with HER2 is clearly similar to the configuration of the combination of ¥£(}17卩王1»(1&quot;.111.8.(1. = 0.7 person, €(1). Two 13111?&amp; 13 structures (^ feet are well superimposed on each other, and with the parent Herceptin® antibody ρν and bH1

Fv(HER2)r.m.s.d.=0.6A, Herceptin® antibody ρν and bHl

Fv(VEGF)r_m.s_d. = 1.2A Good stacking. The CDR-L1 is an exception and the two complex structures are significantly different; the deviation is 4.6 A (residue 27-32 Ca). Loop 30 shows that the CDR conformation of the bHl Fab that binds to VEGF is significantly similar to the bHl and Herceptin® antibodies that bind to hER2, with the exception of CDR-L1. Loop 30 is a superposition of CDR loops as a tube that binds bHl (dark shade) of VEGF, bHl (white) that binds hER2, and a Herceptin® antibody (light shade) that binds to HER2. The CDR-L1 loop exhibited a significantly different configuration of the two bHl structures (r.m.s.d. Ca = 4.6 for bHl residues 27-32) (Figure 31). In the HER2 complex, CDR-L1 is minimally involved in antigen interaction and one part of the loop (residues 28-30b) appears to be flexible. For VEGF binding, the entire loop system is sufficiently structured and accounts for 26% of the surface area embedded by VEGF. The two residues in CDR-L1 (Ile30c and Tyr32) have different configurations and have different roles in the binding of bH1 to HER2 or VEGF. In the HER2 complex, the side chain of Ile30c is embedded in a hydrophobic core formed by CDR-L1 and CDR-L3 residues. In the VEGF complex, the side bond forms a hydrophobic contact with VEGF. The Ca of Tyr32 is in the same position in both structures, but its side chain is rotated by about 130 degrees. In the HER2 complex, Tyr32 overlaps with the receptor, whereas in the VEGF complex, the side chain forms a hydrophobicity with Ile29 142552.doc • 146·201011047 core and supports the configuration of CDR-L1 and CDR-L3. The CDR-L1 configuration is further stabilized by hydrogen bonding between Tyr32 and the LC framework residue Gly72. Structural analysis confirmed that Tyr32 is important for VEGF binding because it is not allowed to mutate to alanine or phenylalanine. In contrast to VEGF binding, Tyr32 mutation to alanine (return to Herceptin® antibody residues) is preferred for HER2 binding. The superposition of the two complexes indicates that VEGF will clash with Tyr32 of CDR-L1 in its HER2 binding state (Fig. 31). In Fig. 31, the side chains of the residues Tyr32, Ile30c, Ile29 and Gly72 are shown as rods. Residues with a temperature factor above the mean are shown in darker shades (residues 28-3 013). The hydrogen bonding between Dings 132 and 〇 1772 is shown by a broken line. • The above results indicate that the ability to rearrange CDR-L1 is bispecific for bHl

It is necessary. Similar conformational flexibility of CDR-L1 has been shown to play a role in antigen recognition by natural antibodies (Jimenez et al, Proc. Natl. Acad. Sci. USA 100: 92, 2003; Mylvaganam et al, J. Mol. Biol. 281:301, 1998). Figures 26, 28, 30, 31 and 32 are generated from crystal structure coordinates using PYMOL (DeLano Scientfic, San Carlos, CA) (PDB code, 3BDY, 3BE1, 1N8Z). The bHl hunting scan was performed to study the antigen binding site of bHIFab, and the phage was used to present the Fab library for hunting and scanning combined mutation induction (Vajdos et al., J. Mol. Biol. 320: 415, 2002; Weiss et al., Proc. Natl. Acad. Sci. USA 97:8950, 2000). Binding selection of antigens (hVEGF and Her2-ECD) to isolate functional lines, followed by dna sequencing allows calculation of the wild-type/mutant ratio at each 142552.doc-147-201011047 change position (Vajdos et al, 2002). These ratios are then used to determine the contribution of each scanned side chain to VEGF and Her2 binding. These results allow for the mapping of functional complements for binding to VEGF and Her2. The bHl hunting library design uses a phage display library to scan for solvent exposed residues in the CDRs, which allow wild type residues to be changed to alanine or wild type (alanine scan) or to homologue residues or wild type (same line) Scan). The nature of the genetic code requires some substitution in the library in addition to Wt/alanine or Wt/homologous residues (Figure 33). Independent heavy and light chain alanine and homolog scanning libraries were constructed. These libraries are shown in Figure 34. The degeneracy ranged from 1.3 xlO5 to 1.3 χΙΟ8 and the actual library size ranged from 6χ109 to 4x101G. Constructing a Hunting Scanning Library As described above, in order to render the bHl Fab on the surface of the Ml 3 phage, the previously described C-designed with the Ml 3 gene-3 minor outer sheath protein was modified using standard molecular biology techniques. The plastid AP2009 of hu4D5 Fab was presented on the phage fused to the terminal domain to encode bHIFab. The C-terminus of the light chain contains an epitope (gD) tag. The "Termination Template" variant of bHl Fab was used as a library template (Sidhu et al., 2004). The light chain alanine and homolog scanning libraries have a stop codon in CDR-L1, CDR-L2 and CDR-L3, and the heavy chain alanine and homologue libraries contain a stop codon in each heavy chain CDR. The library was constructed by the method described previously (Sidhu et al., 2004) using Kunkel mutation induction (Kunkel et al., 1 987) on the respective termination template 142552.doc -148- 201011047. Library Selection A 5 pg/ml capture target (hVEGF109, Her2-ECD or anti-gD mlgG) was applied to a NUNC 96-well Maxisorp immunoculture dish and blocked with 1% BSA (w/v) in PBS. The phage of the above library was propagated with K07 helper phage (NEB) as described (Lee et al., 2004a). The library phage solution is added to the coated plate at a concentration of 1013 phage particles per ml and

Incubate for 1 to 2 hours at room temperature. The plates were washed 8 times with PBST and then the bound phages were lysed with 0.1 M HCl for 30 minutes. The enrichment after each round of selection is determined as previously described. After 2 rounds of target selection, 50 to 1000-fold enrichment was observed for all libraries, and the latter two showed 5 to 10 fold enrichment except for LC-Ala and LC-Hom picked on hVEGF. A large number of random pure lines exhibiting 50 to 1000 fold enrichment in each library were selected for sequencing as described (Sidhu et al., 2004). The library LC-Ala was screened for hVEGF binding by phage ELISA (Sidhu et al., 2000). A pure line that exhibits at least twice the hVEGF ELISA signal over the BSA coated control plate is selected for sequencing. The LC-Hom library was subjected to another round of selection for hVEGF, followed by phage ELISA screening and VEGF binding to the pure line sequence. DNA Sequence Analysis High quality sequences from each of the different target selections were translated and aligned (data not shown). The number of sequences subjected to analysis in each library is summarized in Table 4 below. 142552.doc -149- 201011047 Table 4. Number of Sequences Analyzed Library Total Sequence Number LCA-V2b 51 LCH-V3 79 LCA-H2 97 LCH-H2 50 LCA-gD 112 LCH-gD 120 LCA-pL 60 LCH-pL 65 HCA-V2 100 HCH-V2 96 HCA-H2 81 HCH-H2 96 HCA-gD 105 HCH-gD 105 HCA-pl 102 HCH-pl 99 Calculate the wild-type/mutant ratio at each change position (Figure 35 and Figure 36), thus allowing calculation of the listed F wild type/mutant plastic values (Figures 35 and 36) by dividing the ratio from target selection as described by (Vajdos et al., 2002) The ratio from the presentation selection is corrected for presentation. A s/burma greater than 1 indicates that Wt is better at this position and is less than 1

The important role of F wild type/mutant H tit $ 〇 F wild type/mutant type&gt;5H. The importance of each scanned CDR residue is shown in Figures 37A-37D. The results indicate that residues from both the heavy and light chains actively contribute to the binding of the two antigens (Her2 and hVEGF). The effect of bHl light and heavy chain residues on Her2 binding was compared to the effect of the parent antibody hu4D5 (Kelley and O'Connel 1993) (Fig. 38). Figure 39A and Figure 39B show shotgun alanine scan and homolog scan results for bHl Fab for binding to VEGF and HER2. Alanine mutation (ml) or additional mutations (m2, m3; due to hunting - alanine codon limit 142552.doc -150-201011047) or homologous amino acid mutation (m4) effect in wild type and The mutant (wt/mut) was calculated as the ratio of occurrence in the pure line of human VEGF (Fig. 39A) or HER2 (Fig. 39B). In the case where only wild type residues are present, the ratio is shown to be greater than the "&gt;" wild type count. The identification code for the amino acid substitution (ml-m4) is shown as above the F value. When the wild type residue is alanine, it is substituted with glycine (ml). "*" indicates the degree to which the bH1 residue is embedded after the formation of the VEGF or HER2 complex (*25-49% of the accessible area is embedded, and **50-75% of the accessible area is embedded,** * Greater than or equal to 75% of the _ accessible area is buried).

Residues that significantly contribute to energy interaction constitute a functional paratope that constitutes a subset of structural binding sites. In contrast to the extensive overlap between antigen contact sites, the two functional complementary bits show limited overlap (Figures 32 and 40). In particular, based on shotgun scanning mutations, for VEGF (Fig. 40A) or HER2 (circle 40B) binding, for each mutation pair value of alanine (black bars) or homologous amino acids (white bars) (y-axis, Kcal/mol) mapping. "t" indicates the lower limit, so no mutation was observed at this position. "*" indicates the extent to which the bHl residue surface area is embedded after formation of the VEGF or HER2 complex (*25-49% embedding, **50-75%, ***&gt; 75%). The VEGF binding interaction is dominated by LC CDRs with the CDR-L1 Tyr32 and CDR-L3 His91 as the core hotspots &gt; 1.5 kcal/mol). The HER2 binding is primarily supplied by the HC CDR. Figure 32 shows the crystal structure of bHl and Herceptin® antibody residues based on their functional importance when shaded on the Fab surface (for alanine mutations, dark shades and white text, A^Gkl .5 kcal/mol Moderately shaded and black text, 1"AG&lt;1.5 kcal/mol; light shades and 142552.doc •151- 201011047 black text, 0.5SAAG&lt;1 kcal/mol). As shown in Figure 28, the black dotted line outlines the contact. The outline of the region. The white dotted line shows the separation of the light chain from the heavy chain. For VEGF binding and HER2 binding, the 'functional paratope residue is distributed between HC and LC, which indicates the synergy between the two strands. CDR-H3 Trp95 is the only common hotspot residue of the two interactions (AAGwt/aia > 1.5 kcal/mol). As mentioned above, the 'VEGF binding interaction is mainly mediated by LC CDRs' and the HER2 binding line is composed of HC CDRs. Dominant. Compared to the Herceptin® antibody, bHl with weaker HER2 binding affinity (300-fold) retains the same core hotspot residues (Arg50, Trp95 and TyrlOOa) as HER2, while the importance of the surrounding residues is re-established. Distribution (circle 32). In short, in the heavy chain Most of the important side chains that bind hu4D5/Her2 are still important for bHl/Her2 binding (ΔΔ〇&gt; 1.5 kcal/mol). These are some changes. Light chain residues have more reorganizations in supply - some Residues become less important and some become more important. In summary, the functional sites are part of the structural interface of the crystal structure of the bHl-VEGF and bHl-Her2 complexes. In short, by making the energy mutual A unique set of bHl residues in action interact with each antigen to characterize the interaction of bHl with two structurally unrelated large proteins, although most of the two widely overlapping binding sites of the two different antigens exhibit a single conformation However, the flexibility of one CDR loop (L1) facilitates the accommodation of both HER2 and VEGF. The mechanism is reminiscent of the molecular diversity observed in multiplex-specific antibodies that bind to unrelated small haptens or peptides. Describe the differential localization of small ligands in a spatially distinct region of a single antibody conformation (Sethi et al., Immunity 24: 429, 2006) or multiple pre-presence configurations of the original binding site by anti-142552.doc-152-201011047 (James et al. , Science 299: 1362, 2003) The multiplex specificity mediated by how limited LC mutations can produce antibodies that bind to two unrelated protein antigens further highlights the diversity of antibody molecules in antigen recognition. bHl affinity mature

To investigate whether the VEGF binding affinity of bHl can be increased by optimizing the light chain sequence before structural and functional results become available, construct a library in which the crystal structure based on the h4D542 Fab (assumed to be very similar to bHl Fab) (Eigenbrot) Et al., 2001) Diversification of CDR residues at highly accessible positions in the solvent. The targeted residues were allowed to become wild-type or a few homologous residues (Figure 34). Construct a library as described in the section "Building a Hunting Scanning Library". As described, a solution based selection method is used to select a higher affinity VEGF-conjugate. Perform two rounds of solution based selection. Stringency was increased in each round of selection by reducing the concentration of biotinylated VEGF from 50 nM in the first round to 20 nM in the second round. The 138 pure lines from the last round of selection were sequenced. Most of the pure lines were found to be unique. A high-yield ELISA assay using immobilized VEGF (8-109), anti-gD antibody, and Her2-ECD was used to identify pure lines that bind to VEGF, Her2-ECD, and anti-gD mlgG but not to BSA. The VEGF-ELIS A binding signal was corrected with an anti-gD ELISA signal to estimate the relative affinity of the VEGF-binding pure line. A pure line with a high VEGF/anti-gD ratio was chosen for further characterization. As previously described, the affinities of the selected pure lines for VEGF and Her2 were estimated by competition ELISA as indicated by the phage-presented Fab. The bHl variant showed improved VEGF binding affinity compared to the parental bHl pure line. Interestingly, even though 142552.doc -153- 201011047 does not rely on affinity selection for Her2, some pure lines have a slightly improved IC5 devaluation for Her2 binding. This indicates that it is possible to mature the bHl pure affinity without significantly affecting the Her2 binding ability in terms of VEGF binding. There are some VEGF affinity-improving lines that exhibit reduced Her2 binding affinity compared to the parent bHl pure line. This result indicates that, despite the fact that the heavy chain is a major contributor to binding energy, the light chain is actively contributing to the binding ability of bHl to Her2, based on the bHl-Her2 complex structure and the scanning alanine scanning analysis. The sequence of the characterized pure line and the IC5 enthalpy are summarized in Figure 41. The unique findings of most of the sequences indicate that the light chain sequences of these variants have not been fully optimized for VEGF binding and that it is possible to further improve the bHl pure line by further affinity selection. As shown in Table 5, a significant improvement in the affinity of a single Fab for both antigens is achievable and generally achievable. For example, the KD of human VEGF increased from 250 (bHl; IgG) to 41 (bm-81; IgG) or 16 nM (bHl-44; IgG), and the KD of HER2 increased from 21 (bHl; IgG) to 7 (bHl-81; IgG) or 1 nM (bHl-44; IgG). Affinity is improved by introducing mutations into the HC and LC CDRs of bHl. The location is selected based on information about the functional complements of VEGF and HER2 described herein. The bHl variant was isolated in two steps by selecting and screening the phage display library as described herein. The modified pure line bhl-81 was isolated by selecting the light chain homolog shotgun scanning library based on affinity. In the second step, the highest affinity pure line (bHl-44) was isolated from the library by randomizing the residues of bHl-81. In particular, the oligonucleotides are designed to randomize the points in the HC and LC of bHl-81 (Table 5) to encode approximately 50% of the wild 142552.doc -154- 201011047 at each location. 50% of all other 19 amino acids (Gallop et al, Journal of Medicinal Chimistry 37: 1233, 1994)

The KD of the bH1 affinity-modified variant was measured for the Fab fragment and the IgG antibody (Table 5). Fab fragments and IgG antibodies were visualized in E. coli and 293 cells, respectively, and purified as described herein. The binding affinity of Fab fragments and IgG antibodies was determined using surface acoustic resonance (SPR) measurements using BIAcore 3000 as described in Lee et al. (J. Mol. Biol. 340: 1073, 2004). To study the affinity of antibodies in the form of monovalent Fab fragments, antigens (hVEGF109, murine VEGF102 and HER2 ECD) were immobilized on BIAcore CM5 wafers at low density. Serial dilutions of the Fab fragments were contacted with the immobilized antigen and the binding reaction was measured by SPR measurement. The 1:1 Langmuir combination model was used to calculate tn, hff, and KD. To determine the KD of the IgG antibody, IgG was captured on a BIAcore CM5 wafer by immobilized anti-Fc antibody and exposed to serial dilutions of hVEGF109, murine VEGF102 and HER2-ECD. For HER2, a simple 1:1 Langmuir binding model was used to determine KD, while VEGF required a bivalent analyte model. All experiments were performed at 3 (TC). Table 5 shows the randomized positions in bold and summarizes the CDR sequences of bHl, bHl-81 and bHl-44 (SEQ ID 1: 1-9 and 39-41) and their affinity. (as determined by surface plasma resonance) 142552.doc • 155· 201011047 Table 5. Variants with improved dual affinity bHl anti-hip light chain Kd(nM) HER2 hVEGF mVEGF Fib IgG Fab l^j CDR-LI CDR-L2 CDRL3 33⁄43⁄4衮衾戋 SS S ^ Κ S 3: Κ 5; Si SSS years old bHl 300 250 &gt;1000 i6 21 bHU8] NAKT 58 41 ND ISO 6 7 bH-44 Ν A Κ Τ FSS » 16 33 36 0 7 ' Heavy Chain Resistant CDR-HI CDR-H2 CDR-H3 ras «ss Λ « g 9» « # s * 1 | I ° s bHl bHt-81 bH-44 Ν 1 KDTYSGR 1 VPTNCYTR SEWGCV DGFYAMDV ND = not detected. The monovalent affinity of antibodies to human VEGF109, murine VEGF1〇2 and HER2 ECD was measured by BIAcore. Table 5 shows the representative dissociation constants (Kd) for each binding interaction. Receptor-binding fragment of VEGF (VEGFi〇9) was used, because the bHl variant has a similar affinity in the solution competition experiment. Full-length protein (VEGF165) and VEGF1 () 9 (data not shown). Different assay formats and evaluation models were used to calculate the Kd of Fab fragments/IgG antibodies as described herein. Different assay/evaluation formats produced consistent for individual interactions. Dissociation constants. Example 5. Analysis of IgG activity by cell assay To determine whether bHl and 3-1 antibodies inhibit the proliferation of hVEGF165-induced human umbilical vein endothelial (HUVEC) cells, they were tested for proliferation assays. (Fuh et al. Human umbilical vein endothelial cells (HUVEC) (Cambrex, East Rutherford, NJ) were grown and assayed as described in J_Biol. Chem. 273:11197, 1998. Apply approximately 4000 to each well of a 96-well cell culture dish. HUVECs were incubated for 18 hours in Duss 142552.doc • 156· 201011047 modified Ig's/F-12 medium (1:1) (assay medium) supplemented with 1.0% (v/v) fetal bovine serum. Fresh assay medium with a fixed amount of VEGF (0.2 nM final concentration) is first titrated with a VEGF amount that stimulates sub-DNA synthesis, and then an increased concentration of anti-VEGF antibody (e.g., bHl) is added to the cells. After incubation for 18 hours at 37 ° C, cells were pulsed for 24 hours per well of 5 gCi [3H] thymidine and collected for counting with a TopCount Microplate Scintillation counter. The results indicate that both 3-1 and bHl can inhibit VEGF-induced HUVEC cell growth by preventing hVEGF-induced signaling and subsequent proliferation. Avastin® antibody (anti-VEGF) was used as a positive control and Herceptin® antibody was used as a negative control (Fig. 42).

To study the binding of bispecific anti-Her2/VEGF antibodies to Her2 expressed on mammalian cells, the binding of bHl and bH3 antibodies to NR6 fibroblasts (NR6-Her2) overexpressing Her2 was studied by flow cytometry. . One million NR6-Her2 cells were incubated with 100 pg/ml Fab and IgG for 1 hour and then incubated with Alexa488-conjugated murine anti-human IgG antibody for 1 hour. As a negative control, the binding of Fab and IgG to non-expressing NR6 cells was investigated. As indicated in Figure 43, as Fab and IgG&apos;bH1 and bH3 specifically bind to Her2 on NR6 cells. Figure 44 shows the results of a competitive binding assay of bH1 with VEGF or HER2. Although bH1 is less efficient than Herceptin® antibody due to its reduced affinity, it inhibits VEGF-induced human umbilical vein endothelial cells with IC nig of 200 nM (this is consistent with its affinity of 300 nM) after 5 days of incubation. (HUVEC) proliferation, and inhibition of 142552.doc-157-201011047 proliferation of breast cancer cell line BT474 expressing HER2 (Fig. 45). Herceptin® IgG antibody and bevacizumab (anti-VEGF) were used as controls. As shown in the country 45, the bHl-81 and bHl-44 antibodies inhibited the proliferation of VEGF-induced HUVEC cells and the growth of BT474 cells to a greater extent than bH1. The increase in potency of the bHl variant is related to its relative affinity. The highest affinity variant (bHl-44) inhibited the growth of HUVEC and BT474 cells, respectively, with potency similar to bevacizumab or Herceptin® antibodies. For these experiments, VEGF-stimulated HUVECs were treated with increasing concentrations of human IgG, and proliferation inhibition was measured two days after the incubation as described in Liang et al. (J. Biol. Chem. 281: 951, 2006). Breast cancer cells BT474 were cultured in RPMI medium supplemented with 10% FBS. For the assay, 104 cells were coated in each well of a 96-well culture dish and incubated overnight (18 hours) at 37 °C. An increased concentration of human IgG is added to the cells. The cells were then incubated at 37 °C for five days, followed by the addition of 10% AlamarBlue (Biosource International, Camarillo, CA) according to the manufacturer's instructions. Antibody-dependent inhibition of HER2 expressing cell proliferation was determined after 6 hours by measuring the fluorescent signal. Example 6. Analysis of binding specificity The binding specificity of antibodies derived from LC pools was determined. IgGs bound to various immobilized purified proteins or cell lysates (including homologous antigens) were assayed by ELISA. The antigen was fixed and incubated with hlgG at a concentration of 15 pg/mL for one hour. The bound IgG was detected spectrophotometrically (light density at 450 nm; y-axis; Figure 46). The proteins included in the assay are (from left to right in Figure 46): vascular endothelial growth factor A (VEGF), murine vascular endothelial growth factor 142552.doc -158- 201011047 (murine VEGF), vascular endothelial growth factor C (hVEGF-C), vascular endothelial growth factor D (hVEGF-D), HER2 extracellular domain (HER2 ECD), epidermal growth factor receptor extracellular domain (hEGFR), ErbB3/HER3 extracellular domain (HER3 ECD), human death Body 5 (hDR5), bovine serum albumin (BSA), casein, fetal bovine serum (FBS), neutral streptavidin, 5% milk, mouse fibroblast lysate, and added hVEGF-A or Mouse fibroblast lysate of HER2 ECD. In Fig. 46, the error bars represent the average of the standard errors (SEM) of the two iterations. Untested antibodies bH3, 3-1, bD1, bD2, 4-1, and 4-5 with murine VEGF, HER3 ECD, neutravidin, 5% milk, cell lysate supplemented with hVEGF-A And the binding of cell lysates added with HER2 ECD.

Various antibodies (Avastin® antibody, Herceptin® antibody, bH1, bH3, bH4, bHl-81 and bHl-44) were also assayed to block the ability of VEGF to bind to VEGF receptors (Fig. 47). Biotinylated human VEGF165 (Fig. 47A) or murine VEGF164 (Fig. 47B) was equilibrated with increasing concentrations of IgG (x-axis). Unbound VEGF was captured on the immobilized human VEGFR2-ECD Fc fusion protein and detected spectrophotometrically (optical density at 450 nm, y-axis). Similar inhibition of VEGFR1 was also observed. Anti-VEGF antibodies block the binding of VEGF to the VEGF receptor. The antigens VEGF and HER2 were shown to compete for binding to the bHl-44 bispecific IgG antibody (circle 48) in solution. A human bHl-44 IgG antibody at a concentration of 0.1 nM was incubated with 0.1 nM biotinylated human VEGF!65 in the presence of increasing concentrations of HER2 ECD. 142552.doc -159· 201011047 bHl-44 was captured by immobilized anti-human Fc and bHl-44-conjugated biotin-VEGF detected by streptavidin-HRP. A murine anti-HER2 antibody that binds to a non-overlapping epitope on HER2 was used, followed by HRP-conjugated goat anti-mouse IgG to detect HER2 ECD in combination with captured bHb44 (Fig. 48A). Human bHl-44 IgG at a concentration of 0.2 nM was incubated with 0.6 nM biotinylated HER2 in the presence of increasing concentrations of human VEGF165. bHl-44 was captured by immobilized anti-human Fc and bHl-44-conjugated biotin-HER2 detected by streptavidin-HRP (Fig. 48B). The specific binding of bHl and bHl-44 to cells was also tested by FACS (Fluorescence Activated Cell Sorting; Figure 49). Bispecific antibodies (bH1 and bHl-44) bind to mouse fibroblast (NR6) cells expressing HER2 (Fig. 49B) but not to HER2 negative NR6 cells (Fig. 49A). 500,000 to 1 million cells were incubated with 15 pg/mL hlgG on ice for one hour. Primary antibody bound to cells was detected using a secondary fluorescent PE-conjugated goat anti-human IgG. Cells were analyzed using a FACS Calibur flow cytometer. bHl and 匕1'11-44 did not cross-react with 11£112 rat orthologs because no mouse fibroblasts transfected with rat neu were detected (the rats of HER2 were directly The combination of the source). To further characterize the specificity of the bHl antibody variants bHl-81 and bHl-44, immunoprecipitation experiments were performed and it was shown that bHl antibody variants specifically lyse VEGF or HER2 but not other proteins from mouse fibroblasts (NR6) The substance was immunoprecipitated (Fig. 50). NR6 cells were labeled with non-specific biotin, lysed and solubilized by cell membrane protein detergent. NR6 cells corresponding to 5 million to 10 million cells per ml, NR6 cells supplemented with 0.1 pg/mL biotinylated VEGFi65 or NR6 cells overexpressing 142552.doc-160-201011047 HER2 The cytoplasm was incubated with 15 pg/mL antibody. The protein A coated agarose beads were used to capture the antibody and dissolve the bound protein. The solubilized protein was separated by SDS-PAGE. Cell lysates corresponding to about 25 to 50,000 cells and immunoprecipitates from about 120,000 to 250,000 cells are loaded onto the gel. The captured biotin-labeled protein was detected by Western blotting using the anti-protein _HRP. Example 7. Analysis of IgG activity by in vivo assay To assess whether the in vitro dual activity of such antibodies can be converted to the corresponding in vivo activity, we have used a known anti-VEGF antibody (C〇1〇205' a colorectal cancer cell line. Or a Herceptin® antibody (BT474M1, breast cancer cell line) to treat a reactive mouse xenograft tumor model. In particular, C〇1〇205 xenografts were used in nu/nu mice and BT474M1 xenografts were used in beige nude XID mice. All animal studies were in accordance with the American Association for Accreditation of Laboratory Animal Care and Genentech's Institutional Animal Care and Use Committee. Specifically, BT474M1 (internal) and C〇1〇205 (ATCC, Manassas, VA) cells were cultured in RPMI medium/10% fetal bovine serum. 5χ106 BT474M1 cells suspended in a mixture of Hank's Buffered Salt Solution (HBSS) and matrigel (l:l) were injected into the Harlan beige nude implanted subcutaneously into the female disaccharide pellet. XID mice (Indianapolis, IN) in the breast fat pad. For C〇1〇205 xenografts 142552.doc -161- 201011047, 5χ106 C〇1〇205 cells in HBSS were injected subcutaneously into Charles River nu/nu mice (Hollister, CA). When the average tumor size reached approximately 200 mm3, the mice were randomized into 7 groups of 8 mice (BT474M1) or 10 mice (C〇1〇205). The antibody is administered intraperitoneally once a week. Tumor size was measured twice a week. The volume is calculated from V = 0.5 ab2 (a is the longest dimension of the tumor and b is perpendicular to a). Statistical evaluation was performed using a one-factor analysis followed by a two-tailed student i test. The adjustment of the amount of alpha due to multiple comparisons (Bonferroni) did not change the meaning of our conclusions. The partial response (PR) system is defined as a response in which the tumor volume is reduced by 50% to 99% compared to V0. Serum samples were collected 7 days after the first and third treatments. The concentration of human antibodies was determined using ELISA. The donkey anti-human IgG Fc was immobilized on an immunoculture dish. The serum and antibody standard dilutions were incubated on the plates for 2 hours. The bound antibody was detected by binding to horseradish peroxidase goat anti-human IgG Fc followed by TMB receptor/1M phosphate. The plate was read at 450/620 nm. The sample concentration was determined using a 4-parameter algorithm. The bHl-44 treatment group was treated with anti-VEGF (B20-4.1) (Liang et al, J. Biol. Chem. 28 1:95 1, 2006), Herceptin® antibody or combination (Herceptin® antibody + anti-VEGF) The groups were compared to further confirm that bHl-44 antibody is capable of inhibiting VEGF and HER2-mediated tumor growth. In all groups, antibodies were present at high levels (evaluated by ELIS A) in serum from C〇1〇205 xenografts 7 days after the start of treatment, indicating normal pharmacokinetics (Table 6). 142552.doc -162- 201011047 Table 6. Antibody serum content group antibody concentration (pg/ml) mean SD control group IgG 10 mg/kg 65 14 Herceptin® 10 mg/kg 83 47 anti-VEGF 10 mg/kg 20 8 anti- VEGF+Herceptin® 10+10 mg/kg 41 25 bHl-44 10 mg/kg 30 12 bHl-44 20 mg/kg 37 9 For each group, n=5; SD=standard deviation··

bHl-44 administered at 10 mg/kg per week inhibited C〇1〇205 tumor growth (p&lt;0.0001, n=10) compared to the control antibody, and its efficacy was similar to that of anti-VEGF (10 mg/kg per week). The Herceptin® antibody had no effect on the growth of C〇1〇205 (p=0.12, n=10). As expected, combination therapy showed efficacy similar to anti-VEGF alone. The bm-44 antibody administered at 10 and 20 mg/kg per week produced a dose-dependent response. In the BT474M1 model, observation was observed in mice treated with bHl-44 antibody (10 mg/kg per week, p=0.0005, n=8 and 20 mg/kg per week, p=0.0001, n=7). Significant tumor growth inhibition. As with the combination of Herceptin® antibody or Herceptin®/anti-VEGF combination, more than half of the tumors treated with the bHl-44 antibody showed more than 50% regression from the initial volume (i.e., partial response, Figure 51). On the other hand, anti-VEGF alone showed only a modest growth inhibitory effect on BT474M1 compared to the control group (p = 0.06, n = 7), and showed no partial response. This demonstrates that bispecific bHl-44 antibodies inhibit two different mechanisms important for tumor growth in vivo. The above results indicate that the affinity-modified variants of the bHl antibody (e.g., bHl-44 and bHl-81) inhibit the potential of two mechanisms important for tumor growth in vivo 142552.doc • 163- 201011047. Example 8. Characterization of the binding interface of VEGF and HER2舆bH1 and bHl-44 To further compare the structural features of bHl and bm-44, the binding interfaces of VEGF and HER2 to these antibodies were identified. The structural contacts listed in Table 7 are identified based on the crystal structure coordinates 3BDY(bHl/VEGF) and 3BEl(bHl/HER2). Use the program XSAE to calculate the combined interface. The program defines interfaces that are polar, hydrophobic, and hybrid. Table 7 lists bH1 residues that are embedded in the total surface area of &gt; 25% after HER2 or VEGF binding. Table 7 also lists VEGF and HER2 residues within 4.5 A of the bH1 residue. Based on the crystal ©© structural coordinates 3BDY, 3BE1 and 1N8Z (PDB), the surface area of each residue that was embedded after the formation of the complex was calculated using IMOL. The polar and hydrophobic interface regions reported in Table 11 reflect one of the polar interface regions and the hybrid type. The hydrophobic interface region reported consists of a hydrophobic region and a mixed half. Crystal structure and alanine scanning showed that bHl retained the same binding epitope as Herceptin® antibody on HER2 (Bostrom et al.

2009). The crystal structure of Hereeptin® Fab complexed with HER2 was well superposed on the bHl/HER2 complex (0.8 A r.m.s.d) (Bostrom et al., 2009; Cho et al., 2003). In addition, more than 10% of Herceptin® antibody residues that contribute to total binding energy based on alanine scanning mutations are retained, and many of them are also part of the binding hotspots of bHl and bHl-44 (Bostrom et al., 2009; Kelley and O' Connell, 1993) (Table 14, circle 62). The interface between bHl/VEGF and bHl/HER2 was embedded in 1506 A2 and 1579 A2, respectively, and was mainly hydrophobic (60°/. and 63%, respectively). Herceptin®/HER2 142552.doc •164· 201011047 The binding interface has a similar size and composition to the bHl/HER2 interface (1524 A2, 60% hydrophobicity, Table 11) and is also characterized by high shape complementarity (Table 8). (Bostrom et al., 2009). · Table 7. Structural contact list of bHl Fab/HER2 ECD and bHl/Fab/VEGFi〇9 complexes. The table lists residues that are embedded in &gt; 25% of the total surface area after HER2 or VEGF binding. List the VEGF and HER2 residues within 4.5 A of the bHl residue. Based on the crystal structure coordinates 3BDY, 3BE1, and 1N8Z (PDB), the surface area of each residue that was embedded after the formation of the composite was calculated using IMOL. Area of bHl residue embedded by HER2 (%&gt; Area of HER2 residue exposed to bHl VEGF-embedded area (%) Contacted VEGF residue of bHl Υ33 48 E558 F573 87 H86 R50 97 E558 D560 F573 35 H86 Υ52 30 H86 Υ 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 44 P603 C604 90 P571 P572 55 K569 P571 68 D560 P572 A600 C601 Q602 P605 P572 G92 E93 H90 191 G92 191 Q89 H90 191 H90 M81 Q89 M18 191 Q89 K84 G88 Q89 H90 Q87 G88 087 As described (Lawrence et al., 1993) The shape complementarity between the antibody and the antigen (as indicated by Sc in Table 8). Similar to the complementarity between the Herceptin® antibody and HER2, the high shape complementarity of the bHl/VEGF and bHl/HER2 complexes is Reported antibody-antigen complexes (Sc is about 0.64 to 0.68; Lawrence et al., 1993). HE The superposition of R2 and bHl in its VEGF binding conformation or the superposition of VEGF and bHl in its HER2 binding form • 165-142552.doc 201011047 reveals that the shape complementarity observed when the antibody is juxtaposed with an unrelated antigen is small (Sc It is about 0.35; Lawrence et al., 1993). These results indicate that bH1 rearranges to accommodate two different antigens. Table 8. bHl binding to different surface configurations of HER2 and VEGF. Antibody/antigen complex shape complementation Sex antibody antigen Sc* Herceptin HER2 0.75 bHl HER2 0.72 bHl VEGF 0.68 bHl (configuration of binding to VEGF) HER2 0.40 bHl (configuration of binding to HER2) VEGF 0.44 *Sc = median shape complementarity statistics by bHl The phage display antibody library selects the high affinity variant bHl-44 to improve the affinity of bHl. Hunting for alanine scanning mutation induction indicated that bHl-44 retained the antigen binding hotspot of bHl (Tables 9A-B, 10 and 14). The technique described above for the bHl shotgun alanine scanning mutation was used to induce bHl-44 hunting for alanine scanning mutation induction. In Tables 9A-B, the effects of alanine mutation (ml) or additional mutations (m2, m3; due to the limitations of the shotgun codon) or homologous amino acid mutations (m4) are VEGF (Table 9A) or HER2 ( Table 9B) was calculated in conjunction with the appearance of wild type (wt) in the pure line or VEGF (Table 9A) or HER2 (Table 9B) in combination with the wild type/mutant type of the pure line. When wt is alanine, it is substituted with glycine (ml). For protein folding/expression effects, the wild type/mutant ratio was corrected by dividing by the wild type/mutant ratio from the selection to obtain the F value. The selection is presented independently by selecting a pure line that binds to protein L, which binds to the non-linear epitope of the antibody light chain. When only the Fab heavy chain is fused to the phage in vitro prion protein (p3), protein L binding indicates proper folding and association of the light and heavy chains 142552.doc-166-201011047. In Table 10, the antibody residues which contact bH1 and bH1-44 of VEGF and/or HER2 in the crystal structure are listed. The combined energy hotspot is defined by antibody residues that produce greater than about 10% of the total interaction binding energy of ΔΔGwtMa. The data in Table 11 indicates that the polarity and size of the binding interface are similar between bjji/VEGF, bHl/HER2 and Herceptin®/HER2 complexes. Use XSAE to analyze the polarity of each interface. All numbers shown in Table 11 indicate the area in A2 unless otherwise indicated. Table 9 shows the results of shotgun alanine scanning and homologous scanning of bHl-44 Fab for binding to VEGF. Antigen selection (VEGF) presents selection (an white L} value AAGm«/x«s(kcal/mol) wild wild wild wild wild wild wild F wild F wild F wild F wild type / spondyl / protuberance / sudden Type/protrusion/protrusion/protrusion/protrusion/sinusoidal/protrusion/protrusion/mutation 1 variant 2 variant 3 variant 4 variant 1 variant 2 variant 3 variant 4 辔 jujube 1 variant 2 variant 3 variant 4 /x**· /x**2 */ «Fantasy CDR-Ll §lI29A30K30iiiG31Y32 0.3 1.0 9.0 1.2 0.8 1.8 0.6 3.5 1.2 0'6 39.0 39.0 3.9 36.0 0.8 2.8 ΝΑ 1.7 1.7 0.4 3.0 2.7 9.0 1.6 1.4 7.0 ΝΑ ΝΑ 1.4 0.9 16.0 5.3 0.5 0.9 2.3 15.7 ΝΑ ΝΑ 74.0 1.7 24.0 ΝΑ ΝΑ 74.0 2.6 46.0 23.0 46.0 74.0 0.5 0,9 0.9 0.7 ΝΑ 3.1 ΝΑ 2.1 ΝΑ ΝΑ 1.9 0.4 10 0.4 ΝΑ 1.1 ΝΑ 0.7 ΝΑ ΝΑ 0.3 2.8 8.4 0.8 0.20 .2 0.51.0 0.5 3.01.6 0.4 2.8 46.8 6.5 2.1 7.7 7.1 9.1 9.4 99.1 1.1 0.7 59.8 0.9 2.6 12.4 20.3 0.5 -0.6 3.5 0.1 0.6 8.8 42.7 2.3 9.9 11 7.8 8.7 0.4 2.7 1.2 0.7 0.9 1.2 150.4 1.3 24.3 1.3 152.2 45.9 2.7 0.04 • 0.2 2.4 -0.1 0.6 1.5 1.8 0.7 1.3 1.2 -0.2 3.0 -0.5 -1.1 2.2 1.4 1.3 0.6 -0. 1 3.0 1.9 2.3 CDR-L2 CDR-L3 W50 G51 S52 F53 H91 Y92 593 594 46.0 15.3 46.0 74.0 2.3 1.5 2.4 1.1 20.4 10.2 19.2 65.1 1.8 1.4 1.7 2.5 24.0 ΝΑ ΝΑ 74.0 7.3 ΝΑ ΝΑ 7.5 3.3 9.8 0.7 1.4 15.7 ΝΑ ΝΑ 36.0 4.1 ΝΑ 7.5 7.5 3.9 4.8 0.8 0.9 22.0 44.0 14.7 5.7 1.9 2.4 1.4 0.4 11.6 18.1 10.8 13.5 1.5 1.7 1.4 1.5 7.3 44.0 44.0 73-0 0.0 0.1 0.3 2.6 150.3 880.0 154.0 27.9 3.0 4.0 3.0 48.0 48.0 48.0 13.8 3.8 6.3 1.0 2.8 12.6 7.6 46.7 5.0 1.5 1.2 2.3 1.0 1.0 ΝΑ 1.7 1.7 3.0 ΝΑ ΝΑ 2.5 0.4 0.7 -0.6 -0.2 15.7 ΝΑ 3.4 3.4 1.0 ΝΑ ΝΑ 0.9 15.3 3.7 1.6 0.8 CDR-H1 S30 G31 Τ32 Υ33 ·2·4·50· 12 0 1 ναναναξ NANANA2'2 ·2·2·9·4 5 ·7·6·52· 15 0 1 NANANA2.0 AAA 8 NNN a ·3·°6 1-5·0· ο ο I2· 5 °· 3*~ΐ·9 0.00.4-0.41.2 4 ·°·

2 1 \Λ ο °-a-°L CDR-H2 9 AA 5 8 a30NN6-L -6·8·4·2·2·5 ο 1 1 1 9 1 RS0V52S53E54Y56R58 20.5 0.4 2.1 1.3 70.0 1.3 0.3 0.7 0.3 0.3 -0.7 -0.2 -0.7 -0.8 7.5 1.4 2.0 2.5 1.8 2.7 0.9 11.9 4.3 0.5 -0.1 1.5 0.9 -0.4 ΝΑ 1.0 1.2 ΝΑ ΝΑ 1.1 U 0.9 0.1 0.0 ΝΑ 0.7 0.4 ΝΑ ΝΑ 1.0 3.4 0.7 0.7 •0.3 6.9 0.7 1.6 2.3 1.3 1.2 5.8 2.8 5.3 0.6 1.0 0.6 】.0 -0.3 8.1 1.3 2.1 2.3 4.9 2.7 0.7 0.8 1.6 0.5 -0.2 •0,2 0.3 •0.4 •167- 142552.doc 201011047 W95 139.0 139.0 8.7 V96 G97 0.5 NA NA 0.8 NA NA V98 2.3 NA NA G99 2.1 NA NA F100 6.2 9.5 5.0 YlOOa 27.2 27.2 15.1 ·5-5-7'8·8·8·9 4-15-°'1 1°· 3 AAAA 9 6 °·Ν NN Να0· 2 AAAA ο 5 °-NN NN2·1 09^05^^-0^ °·2·°·1 12-1 -9-2·0·2·2 2· 1· 3· 2· 2·

185.3 685.7 27.2 53.7 3.1 3.9 2.0 2.4 0.2 1.0 -0.9 0.0 0.9 0.5 -0.1 -0.4 1.6 1.4 0.3 0.2 1.7 1.7 0.3 0.3 3.0 4.7 5.4 1.2 0.7 0.9 1.0 0.1 17.9 18.6 26.6 2.5 1.7 1.7 1.9 0.5 ΝΑ == Does not include the mutation table 9. In the case of binding to HER2, the bHl-44 Fab was hunted for alanine scanning and homologous scanning results. Antigen selection (H£R2) presentation choice (Spring White L&gt;

AAGif«.«/3t*c(kcal/mol) F wild ΔΔΟ»? MG wild wild wild wild wild wild wild F wild (10) pro (tw (/h wild MG» Μϋ ΛΛ ΛΛρ type / sudden type / protruding type / sudden Type/protrusion/protrusion/protrusion/protrusion/protrusion/protrusion/protrusion (10)* Variant 丨 variant 2 玺 变 3 variant 4 variant 1 variant 2 variant 3 variant 4 variant 丨 mutant 2 mutant 3 variant 4 1 i «丨丨...咖«/寅》4ί3 /***4 Cdr-li CDR-L2 Q27 0.9 1.0 0.6 1.4 0.8 0.9 0.4 2.8 1.2 1.0 1.3 0.5 0.1 0.0 0.1- 0.4. - N28 1.3 0.9 1.2 U 0.6 0.9 1.0 8.4 2.0 1.0 1.2 0.1 0.4 0.0 0.1 -1-1.2 129 1.5 1.9 0.8 0.8 0.8 0.7 0.4 0.8 1.8 3,0 1.7 1.0 0.3 0.6 0.3 0.0 A30 0.3 NA HA U 0.4 NA NA 0.2 0.8 6.5 -0.2 1.1 | K30a 1.5 2.6 1.8 1.1 1.4 3.1 1.1 02 1.1 0.8 1.6 6.0 0.0 -0.1 0.3 1.1 &quot; T30b 1.0 NA NA 0.3 0.9 NA NA 0.5 1.1 0.6 0.1 -0.3 130c 4.4 5.3 1.8 1.5 2.3 2-1 0.7 1.0 1.9 2.6 2.6 1.6 0.4 0.6 0.6 0.3 S30d 0.9 NA NA 1.0 1.7 NA NA 0.5 0.5 2.1 -0.4 0.4 G31 2.0 NA NA 2.2 2.6 NA NA 3.0 0.8 0.7 -0.1 -0.2 Y32 0.0 1.0 0.02 1.9 0.5 1.9 0.3 1.6 0.1 0.5 0.1 1.2 -1.8 -0.4 -1.6 0.1 W50 81 24.3 8.1 91.0 2.3 1.5 2.4 1.1 3.6 16.2 3.4 80.1 0.8 1.7 0.7 2.6 G51 8.4 NA NA 44.5 7.3 NA NA 7.5 1.2 5.9 0.1 1.1 S52 8.4 NA NA 6.6 4.1 NA NA 7.5 2.1 0.9 0.4 0.1 3.1 9.8 2.0 0.4 1.9 2.4 0.4 1.6 40 0.3 0.8 0.2 CDR-L3 H91 1.7 58.0 58.0 Y92 22.5 90.0 90.0 S93 1.5 NA NA S94 30.3 NA NA 5 18 3 5· 4 1 3 AA °·6·ΝΝ 5 o'· 8'0'0 0·3·3'1' 0.3 2.6 34.0 1160.0 203.0 2.1 2.1 4.2 3.1 0.4 1.0 2.8 5.9 14.2 87.6 1.5 1.1 1.6 2.7 0.2 NA 2.5 0.5 1.1 -0.4 0.1 NA 0.9 29.7 8.1 2.0 1.2 CDR-H1 S30 1.3 NA NA G31 1.5 NA NA T32 0.6 NA NA Y33 150.0 150.0 150.0 ·0·3·5·7 13 15 ·2·2·9'4 NANANA2'0 AAA 8 NNN a -0·3·704· 75.0 0.8 0.0 -0.1 0.7 0.1 -0.2 2.6 -0.2 0.6 179.3 2.5 2.8 2.6 3.1 0.5 c

0 2 3 4 6 8 5 5^^55 RY ^ £ YR ( ^ 0 7 50·0·0·29·3· 1 11 n 11 150.0 150.0 134.0 2.1 1.5 0.9 1.2 2.0 NA NA 1.1 1.2 NA NA 2.2 0.4 147.0 147.0 0.9 1.6 142.0 142.0 66.0 2.1 3 3 2·2·

'2-NN 7 2 2 a·5·9·411 7 0 0 3 3 1 J cj 2-7 12-1 1 I2· °·0·8^ recognition·3·9 7 1 N- x^- 1 4 J13.6 2.1 100.5 2.5 2.8 0.5 2.7 0.6 0.5 0.4 -0.4 -0.3 -0.4 •0.5 1.0 -0.1 0.0 2.2 0.7 0.5 64.1 112.3 0.7 2.0 2.5 2.8 -0.2 61.4 28.8 24.8 1.4 2.4 2.0 1.9 W95 150.0 150.0 150.0 134.0 0.8 V96 0.9 NA NA 1.2 2.1 G97 1.8 NA NA 6.9 0.9 CDR-H3 V9H 0.6 NA NA 1.5 1.5 G99 6.5 NA NA 21.3 1.2 F100 145.0 145.0 29.0 133.0 2.0 YlOOa 149.0 149.0 149.0 6.9 1.5 -0 o. 1 og·4·0·4 ·218 2 0 2 0 5 7 9 5·5·78·88·9 4·1·5·°·1 1°· 3 AAAA 9 I a NNNN aa 740.0 470.0 29.6 3.1 3.9 3.6 2.0 0.8 -0.5 •0.1 1.2 0.4 0.1 1.8 -0.5 0.3 12.0 1.0 1.5 71.1 31.3 73.0 2.5 2.5 2.0 2.5 101.9 262.7 7.8 2.7 2.7 3.3 1,2 ΝΑ = No mutation is included. 142552.doc 168· 201011047 ·· % Table 10. Structural and functional complements of VEGF and HER2. Only VEGF is shared only by HER2 (me 邂邾趄 皱 LC-S30b HC-Y56 LC-Y32 LC-DOc HC-R58 LC-W50 LC-S30d LC-Y53 LC-G31 LC-H91 LC-T93 LC-Y92 LC- T94 HC-Y33 HC-R50 HC-W95 HC-G99 HC-Y100a LC-I29 LC-T94 LC-W50 LC-S30b HC-Y33 HC-W95 LC-S30d HC-R50 LC-G31 HC-Y56 LC-Y32 HC -R58 LC-G51 HC-G99 LC-H91 HC-F100 LC-Y92 HC-YlOOa LC-I29 HC-Y33 LC-H91 LC-T30b HC-R50 LC-S94 LC-S30d HC-Y56 HC-W95 LC-G31 HC-R58 LC-Y32 HC-F100 LC-W50 LC-F53 LC-Y92 HC-YlOOa HC-YlOOa Table 11 · Polarity and size of the binding interface of bHl/VEGF, bHl/HER2 and Herceptin®/HER2 complexes bHl Fab/VEGF binding interface bHl Fab/HER2 binding interface cytidine Fab/HER2 binding interface bHl VEGF combination percentage (%) bHl HER2 combination percentage 赭 赭 HER HER HER HER HER HER HER HER HER HER HER HER HER 311 311 311 311 311 311 311 311 311 295 607 40% 308 282 591 37% 307 308 614 40% Hydrophobic 438 462 900 60% 470 518 988 63% 441 469 910 60% Total 749 757 1506 779 800 1579 747 777 1524 HER2/VEGF dual specific bHl-44 anti-Herceptin® antibody HER2 binding kinetics 142552.doc -169 - 201011047 Perform surface plasmon resonance analysis to study the binding kinetics of bHl and its Fab variants to immobilized VEGF or HER2 (Table 12). Perform a 3卩11 based assay using BIAcore 3000 to allow for a range of 50-150 101 Density of 1111 乂 VEGF109 and HER2 extracellular domains were immobilized on CM5 wafers. Serial dilutions of Fab in PBS with 0.05% Tween 20 were injected at 30 μΐ/min. The binding reaction was corrected by subtracting the reaction of the blank flow cells and by correcting for the buffer effect. The 1:1 Langmuir fitting model was used to estimate ka (association rate) and kd (dissociation rate). The KD value is obtained from the ratio of ka to kd. The bHl Fab/VEGF interaction is characterized by a relatively high association rate (kon = 3.7 x 104) and a fast dissociation rate (kcff = 0.013), which yields a medium nD of 300 nM. The affinity of the bHl/HER2 interaction (Kd=26 nM, kon=9.6x104, koff=2.4xl·3) is a Herceptin positive/HER2 interaction with a slower association rate and a faster dissociation rate (Kd = 0.5 nM) , kon=7.1xl05 'koff=3.5xl(T4) is 52 times lower. The affinity-modified bHl variants (bHl-81 and bHl-44) show an improved association rate and dissociation rate of VEGF and HER2 interactions. The high affinity pure line bHl-44 binds to HER2 with affinity similar to Herceptin® (KD = 0.2 nM, Table 12). Table 12 shows bHl variants determined by surface plasmon resonance measurement at 30 °C using BIAcore And the kinetic profile of the Herceptin® antibody. In these experiments, the Fab was bound to immobilized VEGF or HER2 and the association rate (ka) and dissociation rate (kd) were determined using a 1:1 Langmuir binding fit model. And dissociation constant (KD). The bHl-44 antibody has a similar kinetic profile and affinity for HER2 as the Herceptin® antibody. Loss of two double mutants binding to VEGF or 142552.doc-170-201011047 HER2 (bHl- 44 I29A + Y32A and bHl-44 R50A + R58A) maintain kinetic profile and affinity for other antigens. Table 12. Dynamics of bHl variants and Herceptin® antibodies.

VEGF1M HER2 ECD ka(l/Ms) kd(l/s) Kn(nM) ka (1/Ms) kd(l/s) K〇(nM) Herceptin® Fab - . NB 7.1E+05 3.5E-04 0.5 +A 0.06 Herceptin®(R50A) Fab - - NB 2.7E+04 2.0E-03 74 Herceptin®(R5 8 A) Fab - - NB 5.9E+04 7.3E-04 12 Herceptin® (R50A+R58A) NB NB Fab bHl Fab 3.7E+04 0.013 300 +/- 87 9.6E+04 2.4E-03 26 +/- 28 bHl-81 1.2E+05 0.007 58+/-12 2.2E+05 1.4E-03 6+ /-0.6 bHl-44 Fab 4.0E+05 0.001 3+/-0.3 3.7E+05 8.0E-05 0.2 +/- 0.07 bHl-44(Y32A)Fab bHl-44(I29A+Y32A) - - Weak 6.2E +05 3.5E-05 0.1 Fab - NB 4.2E+05 8.3E-05 0.2 +/- 0.07 bHl-44 (R50A+R58A) Fab 3.5E+05 0.001 3+/-0.7 - - NB NB = no anger The combination of measurements. Dual-specific antibodies interact with HER2 and VEGF with similar thermodynamic properties. Isothermal titration calorimetry (ITC) was used to determine bH1 Fab variants and two antigens VEGF (receptor binding domain of VEGF' VEGF8-i焓9) and HER2 extracellular domain (ECD) interactions between 焓 (ΔΗ) and entropy (AS) changes (Fig. 59A-F, Fig. 60, Table 13). Microcalorimetric measurements of the interaction between Fab and human VEGF109 and the extracellular domain of HER2 were performed using a VP-ITC titration calorimeter (Microcal Inc.) as described (Starovasnik et al., 1999). The protein solution was extensively dialyzed into phosphate buffered saline. The antigen and Fab are dialyzed in the same container to minimize the mixing heat effect due to differences in buffer composition. Fabs at a concentration of 100-220 μM are titrated to an antigen solution at a concentration of 10-22 μM • 171 - 142552.doc 201011047 (HER2-ECD or VEGF1()9). This concentration of antigen is required for accurate sputum measurement, but excludes the determination of KD with high binding affinity. 15 or 20 injections were performed to obtain a 2-fold excess of antibody. The heat of reaction was measured, the heat of dilution of Fab was subtracted, and ΔΗ was calculated. The binding free energy (AG) was calculated according to the dissociation constant (KD) determined by surface plasma resonance (Table 12) according to the following formula: AG=RT Ιη(ΚΌ) The entropy change after association was calculated according to the following formula (AS) ): AS = (AH - AG) / T, where T is the temperature (K). For the determination of ACp, microcalorimetry was carried out at different temperatures ranging from 20 to 37 °C as described above. ACp was measured by linear regression by plotting ΔΗ as a function of temperature (Fig. 62). The interaction of the dual specific antibody (bHl) with either of its two antigens (VEGF and HER2) is first characterized. The combination of bHl with VEGF and HER2 exhibited similar thermodynamic properties (Table 13). The two interactions measured in PBS (pH 7.4) at 30 °C were exothermic (ΔΗ=-2.4 and -2.4 kcal/mol for VEGF and HER2, respectively, Table 13, Figure 60), It is highly advantageous to contribute to the entropy change of binding energy (-TAS = -6.6 and -7.9 kcal/mol for VEGF and HER2, respectively, Table 13, Figure 60). Table 13 shows AG (binding free energy), AS (entropy change), and ΔΗ (焓 change) in kcal/mol. The affinity shown is measured by kinetic analysis at 30 °C by BIAcore in at least two independent experiments. The ΔΗ system is measured using ITC and it represents the average of two or three independent measurements followed by the standard deviation. Calculate AG and AS as described above. 142552.doc -172- 201011047 The high-affinity variants bHl-81 and bHl-44 display a thermodynamic profile similar to bHl. Its interaction with VEGF and HER2 is also characterized by advantages and entropy (Table 13, Figure 60). For the VEGF interaction, the affinity improvement system is significantly more favorable for 焓 change (at 30 ° C, ΔΗ = -7·1 kcal/mol for bHl-44, and bHl is -2.4 kcal for bHl /mol) and a slightly less positive entropy change (at 30 °C, -TAS = -6.6 for bHl-44, relatively bHl is -4·7, Table 13, Figure 60). The improved affinity for HER2 is also associated with a more favorable ❹ 变化 change (at 30 ° C, ΔΗ = -5 · 3 vs. -2.4 kcal / mo, Table 13, Figure 60). Table 13. Antigen binding affinity and heat of bHl variants and Herceptin® antibodies. Mechanics.

VEGF, 〇9_ _HER2-ECD (nM) AG ΔΗ -TAS (nM) AG AH -TAS Herceptin® - - - 0.5 -12.9+/-0.06 -13.6+/- 0.2 -0.3+/-0.2 bHl 300 -9.0+ /-0.2 -2.4+/-0.7 -6.6+/-0.7 26 -10.5+/- 0.4 -2.4+/-0.5 -7.9+/-0.6 bHl-81 58 -10+/-0.1 -6.2+/-0.1 -3.8+/-0.2 6 -11.4+/-0.05 -3.8 -7.6 bHl-44 3 -11.8+/-0.07 -7.1+/-0.3 -4.7+/-0.3 0.2 -13.5/- 0.3 -5.3/-0.4 -8.1+/-0.5 bHl-44 (LC-I29A/Y32A) - - - - 0.2 -13.5/-0.3 -6.4/-0.5 -7.6+/-0.6 bHl-44 (HCR50A/R58A) 4 -11.6+/ - 0.1 -7.7 -3.9 - - - - bHl-44 and Herceptin® interact with HER2 in a unique thermodynamics. In contrast to dual-specific antibodies, the HER2/Herceptin® interaction is characterized by a large beneficial change (ΔΗ = -13.6 Kcal/mol) without any significant entropy change (-TAS = -0.3 kcal/mol, Figure 60, Table 13) (Kelley et al., 1992). Although bHl-44 interacts with HER2 with a similar affinity to Herceptin®, the binding free energy is dominated by a larger entropy component (-TAS = -8.1 kcal/mol, 30 °C) and a smaller initial component kcal /mol, 142552.doc -173- 201011047 30 °C) composition. The unique thermodynamic properties are in contrast to the many similarities in the HER2 binding characteristics between Herceptin® and bHl-44, which include many residues in affinity, kinetics and energy hotspots. Although more than 10% of the total binding energy for HER2 contributes to the residues of Herceptin®, which are similar to those of bHl and bHl-44, there are still some significant differences. Table 14 shows the bHl, bHl-44 and Herceptin® antibody hot spots measured for HER2 binding by alanine scanning mutation. Mutation induction was performed as described in Kelley et al., 1993. The numbers in Table 14 indicate changes in binding free energy when the residue is mutated to alanine (AAG wild-type*). The hotspot residues in Table 14 ©© are shaded and defined as AAGs greater than or equal to 10% of the total binding free energy (AG). In the sequence b ij of bHl, the residues LC-Thr94, HC-Tyr33, HC-Asp98 are conserved, but have different functions in HER2 binding (Table 14, Figure 61). Thus, mutations in the antigen binding site of Herceptin® that recruits VEGF binding appear to cause some fundamental changes in the antigen binding site that affects the interaction with HER2. Dual-specific antibodies adapt to introduced mutations by exploiting different HER2 recognition strategies that produce HER2 affinity for Herceptin®. Interestingly, it was noted that in addition to LC-Ser94 of bHl-44, mutations that improved the affinity for HER2 by more than 100-fold compared to bHl were not part of the hotspot and seemed to optimize existing interactions. The large negative heat capacity in the dual-specific interaction is a further understanding of the common energetics that promote dual-specific interactions and how they differ from the energetics of the monospecific parent Herceptin®, a 142552.doc -174- 201011047 series Experiments were conducted to study the following three Fab/antigen interactions: bHl-44 with VEGF or HER2 and Herceptin® and HER2. The heat capacity of the dual specific interaction was measured by measuring the bound enthalpy (ΔΗ) at a plurality of temperatures ranging from 20 ° C to 37 ° C (ΔΤ = 17° (:, Fig. 62, Table 15). ACp) is a function of ΔΗ and temperature (T) and is expressed by the following equation: ΔΟρ=δ(ΑΗ)/δΤ. ACp is estimated by linear regression according to the temperature-dependent slope of ΑΗ (Fig. 62, Table 15) For interaction with VEGF, the ACp • b of bHl-44 was determined to be -400 cal/mol K and for the interaction with HER2 was -440 cal/mol K. As previously (Kauzmann, 1959) As stated, the large negative heat capacity indicates the importance of the hydrophobic effect, which is consistent with the hydrophobic nature of the structural interface in the two complexes (Table 11). The Δ Cp of Herceptin®/HER2 (previously measured at similar temperature intervals) _370 cal/mol K (Kelley et al., 1992)) is less than the ACp of bHl-44/HER2, but still indicates an important role for the hydrophobic effect in HER2 binding.

The total entropy change (AS) of the combined free energy is the sum of the entropy changes of the following three sources (Murphy et al., 1994): the entropy change (ASsolv) associated with the desolvation of the binding surface, from the rotational and translational degrees of freedom Entropy change in loss (ΔSRT) and entropy change (ASconf) due to changes in the configuration and configuration dynamics of the interacting molecules. (1) AStot=ASs〇lv+ASrt+ASc〇nf Generally, only ASsolv is positive and both ASrt and ASconf are negative. For the association of two molecules, the term "cratic" entropy ASrt can be estimated to be -8 cal/Kmol as described (Murphy et al., 1994). Since 142552.doc -175· 201011047 is embedded in the non-polar surface area, it can be assumed that AS SOLV is dominated by the hydrophobic effect and can be explained by the function of ACp: (2) ASsolv= ΔΟρ 1η(Τ/Τ*) &gt; Τ*=385 Κ △ Sc〇NF can therefore be estimated as follows. (3) ASconf=AStot - ASrt - ASsolv According to equation (3), ASsolv is estimated to be 96 calmoPK·1 for bHl-44/VEGF, 105 calmoPK1 for bHl-44/HER2 and 89 calmor1!^1 for Herceptin®/HER2 (Table 15). This translates to AS CONF * -72 calmoPK·1 for bHl-44/VEGF, -70 calmol^K-1 for bHl-44/HER2, and -80 calmoPK for Herceptin®/HER2 · 1 (Table 15). To test the overall structural stability of the dual specific Fab compared to its parent Herceptin®, a differential scanning calorimetry (DSC) was used for thermal denaturation experiments. Thermal denaturation experiments were performed on a differential scanning calorimeter from Microcal Inc. The Fab was dialyzed against 10 mM sodium acetate (pH 5), 1 50 mM gasified sodium. The solution was adjusted to a concentration of 0.5 mg/ml and heated to 95 ° C at a rate of 1 ° C/min. Baseline calibration of the melting curve and correction. The melting temperature (TM) is determined using software supplied by the manufacturer. As expected, no Fab exhibited a reversible thermal denaturation profile (Kelley et al., 1992) (data not shown). The TM of the dual specific variant (77.2 ° C, 75.6 ° C, 74.3 ° C for bHl, bHl-81 and bHl_44, Table 16) is slightly lower than the Herceptin® TM (82.5 ° C), but High and in the range of Tm reported for other therapeutic antibodies (Garber and Demarest, 2007) 0 Binding of bHl variants with high affinity for VEGF or HER2 alone 142552.doc -176- 201011047 Mechanics and thermodynamics interesting In contrast, dual specific antibodies derive most of their binding energy from a completely unique region that shares the VEGF/HER2 binding site. These data show that VEGF or HER2 binding function of dual specific antibodies can be selectively disrupted without affecting the remaining binding specificity. Structural studies indicate that the structural complements on bH1 overlap significantly for VEGF and HER2, but the induction of alanine mutations in bHl and bHl-44 suggests that the VEGF and HER2 interactions are composed of two distinct CDR residues with few overlaps. The base is mediated (Figures 54 and 57 'Tables 9A, 9B and 10). bHl and bHl-44 are used to scan for alanine scans indicating that some CDR residues are only important for binding to VEGF or HER2 (Figures 54 and 57, Tables 9A, 9B and 10) 'including LC-Ile29, LC -Tyr32 (which is important for VEGF binding) and HC--Arg50, HC-Arg58 (for HER2 binding) (Figures 54 and 57, Table 9 and

10). To confirm the unique importance of the side chains of these residues in each interaction, bHl-44 (LC-Ile29, LC-Tyr32, HC, Arg50, HC-Arg58) or Herceptin® (HC-Arg50, HC) In the -Arg58) framework, each residue is mutated individually or in combination to alanine and the mutant is expressed as Fab and IgG. A vector encoding bHl-44 or Herceptin® Fab fused to the N-terminus of gene III via a heavy chain was used as a template for Kunkel mutation induction (Kunkel et al., 1987). Oligonucleotides are designed to introduce the desired alanine mutation at the selected position. Fab alanine mutants were visualized as phage and binding was confirmed by competitive ELISA (Figure 58). The heavy and light chain variable domains are then cloned into Fab and IgG expression vectors and Fab and IgG are expressed and purified as described (Bostrom et al., 2009). SDS-PAGE confirmed the correct protein size (Figure 65). Size exclusion chromatography showed a degree of aggregation of less than 5%. 142552.doc -177- 201011047 Binding to both antigens by competitive ELISA and/or BIAcore assay. All single alanine mutations in the bHl-44 architecture weakened the binding to varying degrees (data not shown). The most significant single mutation was LC-Y32A, which significantly disrupted VEGF binding while maintaining HER2 binding affinity and kinetics (Table 12, Figure 58 and circle 63). The double mutations I29A+Y32A (LC) or R50A+R58A (HC) almost completely disrupted binding to VEGF or HER2, respectively, while maintaining binding affinity and kinetics for the other antigen (Table 12, Figure 58 and circle 63). The alanine mutations HC-R50A and HC-R58A in the Herceptin® architecture also disrupted binding to HER2 to varying degrees, while the double mutant HC R50A+R58A showed no detectable HER2 binding (Table 12). The thermodynamic parameters of the double mutants were then analyzed and compared to the values of bHl-44. The binding free energy of bHl-44 mutant LC-I29A+Y32A and HC-R50A+R58A to HER2 or VEGF is a favorable contribution of enthalpy and entropy, respectively (for VEGF, ΔΗ=-7.7 and -TAS = -3.9, For HER2, ΔΗ = -6.4 and -TAS = -7.6, Table 13, circle 60) was generated, which is approximately equal to bm-44 measured at 30 °C (Table 13, circle 60). Thus, the double mutant exhibited the same thermodynamic and kinetic profile as bHl-44. 142552.doc 178- 201011047 Table 14. Comparison of bHl, bHl-44 and Hei*ceptin® hotspots for HER2 binding induced by alanine scanning mutations ___AAG (kcal/mol)___ bHl/VEGF | bHl~44/VEGF bHl/Hi:R2 bHl-44/HER2 Herceptin light chain

··

Heavy chain 30 31 32 33 50 52 51 54 56 58 95 96 97 98 100 100a 0.2 0.2 -0.4 0.3C -0.3c 0.4 -0.5 -0.2 0.0 0.4 -0.4 1.2 -0.7 -0.1 0.1 0.7 -0.3 0.01a 0.6 0.4 0.1 a 0.2 -0.4 -0.2 0.4

-0.8 -0.4 0.2 0.06 -0.07® 0.1 -0.8 0.7a -0.1

a is different from the bHl/bHl-44 residue of the Herceptin® antibody. c indicates the contact residue in the bHl/VEGF or bHl/HER2 complex structure. (a) Indicating that the Herceptin® antibody has no residues at this position. 142552.doc -179- 201011047 Table 15. Thermodynamic parameters of VEGF and HER2 interactions. ACp(cal/Kmol) AStot (cal/Kmol) △Sconf (cal/Kmo) △Sdesolv (cal/Kmol) ASrt(cal/Kmol) bHl-44/VEGF -400 16 -72 96 -8 bHl-44/HER2 -440 27 -70 105 -8 Herceptin®/HER2 -370 0.8 -80 89 -8 △Sconf=AStot-ASsolv-ASrt ' As described by Murphy et al., ZVoie/i, iP. For a simple binding reaction, the ASRT is estimated to be -8 cal/molK. ASSOLV = AS * + M: pln (T / Ts *), where T = 303.15, Ts * = 385.15 and AS * is approximately zero. Table 16. Melting temperatures (Tm) of dual specific Fab and Herceptin® antibodies

Fab Tm(°C) 5 2 6 3 2·7·5·4· 8 7 7 7 Herceptin bHl bHl-81 bHl-44 Structural basis for the function of specifically altered residues Next, analysis with VEGF or HER2 The crystal structure of the composite bH (Bostrom et al., 2009) reveals the specific interaction of the binding determinants in each antigen complex (Figure 64). The resulting analysis explains how two specificity-determining residues of a residue disrupt the ability to bind to another antigen without affecting the affinity, kinetics, and thermodynamics of one antigen. The CDR-L1 of bHl contains most of the changes in the sequence of Herceptin® and is important for Ve〇f binding. The CDR-L1 configuration of bHl was significantly different in the two complex structures; the mean deviation was 4.6 A (residues 27-32 2Ca). In contrast, the overall configuration of the bHl Fab complexed with VEGF is clearly similar to the configuration of the Fab binding to HER2 (r.m.s.d. = 0.7 A, for 398 backbone atoms, Ca). When the CDR-L1 loop is located around the HER2 paratope and minimally involves HER2 contact, the loop occupies 26 of the surface area entrapped by VEGF. /〇. 142552.doc -180- 201011047 The superposition of the two complexes indicates that VEGF will conflict with the adjacent residues of Tyr32 and CDR-L1 in their binding to HER2. The main chain of Ty r3 2 Ca atoms are in the same position in both structures, but their side chains are rotated by about 130°. In the VEGF complex, Tyr32 and Ile29 appear to play a structural role in making possible formation of the CDR-L1 required for VEGF binding. Mutation of Tyr32 to Ala or Phe is not acceptable for VEGF binding (Bostrom et al., 2009). Although the side chain of Tyr32 faces HER2, it does not appear to be involved in the production of antigen-contacting Ile29 away from HER2, the side 眷_chain is exposed to solvent and the mutation of Ile29 and Tyr32 to Ala is fully tolerable for HER2 binding.

The structure of the residue of the bHl/HER2 complex which is uniquely important for HER2 binding was also examined. The side chain of Arg50 and Arg58 in the bHl-HER2 structure overlaps with the acidic residues on HER2 (Glu558 and Asp560) (Fig. 64). The interaction appears to be highly side chain specific due to mutations in Lys and Ala destructive (Bostrom et al., 2009). However, in the VEGF structure, Arg50 and Arg58 are exposed to solvents and away from VEGF, and mutations to the Ala or Lys lines are fully tolerable (Bostrom et al., 2009). The citations are as follows:

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Winn, M. D., Μ. N. Isupov and G. N. Murshudov, 2001, Acta Crystallogr. D. Biol. Crystallogr, 57th edition, page 122. All of the patents, patent applications, patent application publications and other publications cited in the specification are hereby incorporated by reference in their entirety in the entireties in The application, patent application publication or publication is incorporated by reference in its entirety. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the design diversity of various LC libraries; Figure 2 shows an overview of four light chain libraries for altering anti-VEGF antibodies or anti-Her2 antibodies to bind to another standard. The italic NNK and XYZ refer to the codon set. Ys, Ds, Ts and Ss refer to the occurrence of tyrosine, aspartic acid, threonine and serine at 50% and the occurrence of either of the 20 amino acids. 50% soft randomization. D/Ds and T/Ts refer to soft randomization where the probability of occurrence of D or T is 75% and the occurrence of 142552.doc -186- 201011047 is 25% of the other 20 amino acids, respectively. Figure 3 shows the sequence of the HC, LC CDR residues of the light chain template; Figure 4 shows the natural and designal diversity of the light chain CDRs. The Herceptin® antibody sequence at each position is shown in brackets. "*" indicates an insertion that does not exist in the Herceptin® antibody;

Figure 5A and Figures 5B1-5B2 show sequences of specific antigen-binding pure lines isolated from a light chain (LC) library. Figure 5A shows LC CDR sequences of monospecific phage-pure lines that bind to VEGF, DR5 and Fc, and Figure 5B shows bispecific Fabs that bind to VEGF/HER2, DR5/HER2 and Fc/HER2. The light chain framework and heavy chain sequences are identical to Herceptin® antibodies except that the LC framework replaces R66G; Figure 6 is a graph showing the binding specificity of antibodies derived from LC pools. The results of the antibodies bHl, bH3, 3-1, bDl, bD2, 4-1 and 4-5 are shown on the graph. The bound IgG antibody (optical density at 450 nm, y-axis) was detected spectrophotometrically. The proteins included in the assay are (from left to right for each antibody): human vascular endothelial growth factor VIII (1^£0卩-eight), 11¥ ugly 0?-C, hVEGF-D, hHER2 extracellular domain (ECD), epidermal growth factor receptor extracellular domain (hEGFR), human death receptor 5 (hDR5), bovine serum albumin (BSA), casein, fetal bovine serum (FBS), WIL2 cell lysate, and NR6 cells Lysates; Figure 7 shows the sorting conditions and enrichment of libraries C and D; Figure 8 shows VEGF conjugates. Residues 28, 30, 30a, 31, 92, 93 and 93a are completely different. Residues 32, 50, 53, 91 and 94 are defined. Residues 29, 33, and 51 are restricted (&lt;3); 142552.doc • 187· 201011047 Figure 9 shows human VEGF conjugates, combinatorial plates and solution selection; Figures 10A and 10B show both VEGF and HER2 binding Pure line; Figure 11 shows the pure line that binds only VEGF and loses binding activity to HER2; Figure 12 shows the pure line that binds to VEGF; Figure 13A and Figure 13B show the pure line that blocks the binding of VEGF to VEGFR1-D2 or D1; Figure 14A and Figure 14B displays the affinity of the VEGF conjugate and the VEGF conjugate from library L1/L2/L3-C, D; Figure 15 shows the pure line that binds both hVEGF and HER2; Figure 16 shows the formation for scFv'2 and presented in LC library conjugates on phage; Figure 17 shows the performance of various pure lines in the form of Fab or hlgG; Figure 18 and Figure 186 show the ELISA of 111 § 0 pure line in combination with 11¥£0?165;

Figure 19 shows an ELISA of the hlgG-type pure line that binds to the immobilized protein target; Figure 20 shows a competitive ELISA of the hlgG-type pure line in the presence of Her2 and VEGF or DR5; Figure 21 shows a Biacore analysis for binding to VEGF or HER2; Figure 22 shows the binding of IgG or Fab with different light chain from different binding lines to HER2-ECD or hVEGF; Figure 23A and Figure 23B show the resistance to block the interaction of VEGF with VEGFR1 D 1-3 and KDR D1-7 VEGF antibody; 142552.doc • 188· 201011047 Figure 24 shows blocking of B20-4.1 binding to VEGF; Figure 25 shows blocking of Avastin® antibody binding to VEGF; Figure 26 shows bispecific binding to HER2 or VEGF Figure 27 is a diagram showing the anti-VEGF antibody blocking the binding of hVEGF to VEGF receptor 2 (VEGFR2); Figure 28 shows the crystal structure of the bispecific bHl Fab bound to HER2 or VEGF; A series of pie charts that contribute to the individual CDRs of the structural complement of bHl. The complementary size of VEGF is 730 A2 and the complementary size of HER2 is 690 A2. The heavy chain CDRs are indicated in gray and the light chain CDRs are indicated in white; Figure 30 shows the superposition of the CDR loops of VEGF/HER2 binding bhl or HER2 binding Herceptin® antibody in the same orientation as Figure 28;

Figure 31 shows the crystal structure of a bispecific bHl Fab bound to HER2 or VEGF. The CDR-L1 lines of the two bHl complexes are shown in the same orientation; Figure 32 shows the energy-important binding sites for bHl in terms of VEGF and HER2 binding; Figure 33 shows the codons of bHl scanned by shotgun; Figure 34 shows Library consortium; Figure 35 shows an antibody-only line with a shotgun scan mutation screened for binding to VEGF; Figure 36 shows an antibody-only line with a shotgun scan mutation screened for binding to HER2; 142552.doc -189- 201011047 Figures 37A-37D show the results of alanine scanning. Figure 37A and Figure 37B show the results of scanning for bHl alanine for VEGF binding or HER2 binding (Figure 37A, Figure 37B, respectively) and for homologs of bH 1 for VEGF binding or JJER2 binding ( Figure 37C, Figure 37D); Figure 38 shows alanine scan results for bHl or Herceptin® antibody mutants; Figure 39A1-39A3 and Figure 39B1-39B3 show the spray of propylamine to bHl Fab for binding to VEGF and HER2 Acid scanning and homolog scanning results; Figure 40 shows the energy-important binding sites of bH1 for VEGF and HER2 binding; Figure 41 shows bH 1 VEGF affinity matured pure sequence and binding affinity for veGF or HER2; 42 shows the inhibition of VEGF-induced HUVEC cell proliferation by anti-VEGF antibody; Figure 43 shows the binding of bispecific antibody to HER2 expressed on NR6 cells; Figure 44 shows the results of competitive binding experiments of bHl with VEGF or HER2; Figure 45 shows that bH1 and affinity-modified variants bHl-44 and bHl-81 IgG inhibit HER2 and VEGF-mediated cell proliferation in vitro; Figure 46 shows the binding specificity of bispecific antibodies derived from LC pool; Display anti-VEGF resistance Blocking VEGF binding to VEGFR2 receptor. Figure 47A shows human VEGF binding and 囷47B displays murine VEGF binding; Figure 48A and Figure 48B show that VEGF and HER2 compete for binding in solution to 142552.doc-190·201011047 bHl-44 bispecific IgG; Figure 49A and Figure 49B showed that the bispecific antibodies bH1 and bHl-44 bind to HER2 expressing mouse fibroblasts (NR6; Figure 49B) but not to HER2 negative NR6 cells (Fig. 49A); Figure 50 shows bispecific bH1 The antibody specifically immunoprecipitates VEGF or HER2 but not other proteins from mouse fibroblast (NR6) lysate; Figure 51 shows bHl-44 for C〇1〇205 in immunocompromised mice and Tumor Suppressor Effect of BT474M1 Xenografts; Figures 52A, 52B and 53 show exemplary receptor human common framework sequences for carrying out the invention and having the following sequence identifiers: Variable heavy chain (VH) common framework (Figure 52A and Figure 52B) Human VH subpopulation I common framework regions FR1, FR2, FR3 and FR4 lacking Kabat CDRs (IA: SEQ ID NO: 42-45, respectively) Human VH subpopulation I common framework regions FR1, FR2 lacking extended hypervariable regions , %FR3 and FR4 (IB: SEQ ID NO: 46, 47, 44 and 45; 1C: SEQ ID NOs: 46-48 and 45; ID: SEQ ID NO: 42 '47, 49 and 45, respectively) Human VH subgroup II common framework regions FR1, FR2, FR3 and FR4 lacking Kabat CDRs (IIA: The human VH subgroup II common framework regions FR1, FR2, FR3 and FR4 lacking the extended hypervariable region are SEQ ID NOS: 50-52 and 45, respectively (IIB: SEQ ID NO: 53, 54, 52 and 45, respectively; IIC: SEQ ID NOs: 53-55 and 45, respectively; IID: SEQ ID NO: 53, 54, 56 and 45, respectively) 142552.doc -191 - 201011047 Human VH subgroup III common framework region FRl lacking Kabat CDRs , FR2, FR3 and FR4 (IIIA: SEQ ID NOS: 57-59 and 45, respectively) lack of extended hypervariable region of human VH subgroup III common framework regions FR1, FR2, FR3 and FR4 (IIIB: SEQ ID NO, respectively : 60, 61, 59, and 45; 111 (:: 8 £ () 1 〇 > 1 〇: 60-62 and 45; 111 〇: SEQ ID NO: 60, 61, 63, and 45, respectively) The human VH receptor framework regions of the Kabat CDRs, FRi, FR2, FR3, and FR4 (receptor A: SEQ ID NOS: 64, 58, 65, and 45, respectively) lack the human VH receptor framework regions FR1, FR2 that extend the hypervariable region. , FR3 and FR4 (receptor B: SEQ ID NO: 60, 61, 65 and 45, respectively; C: SEQ ID NO: 60, 61 '66 and 45, respectively, human VH receptor 2 framework regions FR1, FR2, FR3 and FR4 lacking Kabat CDRs (second receptor A: SEQ ID NO: 64, 58, respectively , 67 and 45) lack of human VH receptor 2 framework regions FR1, FR2, FR3 and FR4 that extend the hypervariable region (second receptor B: SEQ ID NOs: 60, 61, 67 and 45, respectively; second receptor C: SEQ ID NOS: 60, 61, 68 and 45, respectively; second receptor D: SEQ IDn 〇: 60, 61, 69 and 45) Variable light chain (VL) common framework (Fig. 53) Human VL κ subgroup I common framework regions fri, FR2, FR3 and FR4 (kvl: SEQ ID NO: 70-73, respectively) Human VL·κ subgroup π common framework regions fri, FR2, FR3 and FR4 (kv2: SEQidNO, respectively :74-76 and 73) Human VL κ subgroup III common framework regions fri, FR2 and FR3 (kv3: SEQ ID NOs: 77-79 and 73, respectively) 142552.doc 192· 201011047 Human VL κ subgroup IV common framework Regions FR1, FR2, and FR3 (kv4: SEQ ID NOS: 80-82 and 73, respectively); Figure 54 shows residues that form structural or energy interactions with HER2, VEGF, or both. Residues that result in structural contact (&gt;25% embedding) or energy interaction (ΔΑΟ&gt; 10% total binding energy) with HER2 (light grey), VEGF (gray), or both (shared, black) Binding to the surface of bHl of HER2; Figure 55 shows the bHl/VEGF and bHl/HER2 binding interfaces. A close-up of the bHl/VEGF (A) and bHl/HER2 (B) binding interfaces illustrates the structural differences between VEGF and HER2 in the antibody binding region. The surface representations of VEGF (C) and HER2-ECD (D) are shown in the same orientation relative to bHl Fab. Residues that are in contact with bHl Fab (nearly 4. 5 A) are highlighted. In terms of chemical composition or topology, there is no significant similarity between the two epitopes of bHl;

Figure 56 shows that bHl and bHl-44 antibodies block human VEGF binding to VEGFRl. Biotinylated human VEGF165 with increasing concentration (X-axis)

IgG-cultured, then captured on immobilized human VEGFR1-Fc, and detected with streptavidin combined with horseradish peroxidase with added substrate (corrected % OD45G, y-axis); 57 shows the results of alanine scanning of bHl and bHl-44 mutants. Alanine scanning mutations induce the identification of residues that are functionally important for VEGF and/or HER2 binding. The F value indicates the relative contribution of each scanned residue to antigen binding. The F value (black bars) was determined for the binding of bHl-44 to VEGF and HER2 and compared to the F value of bHl (white bars). The amino acid in brackets indicates 142552.doc •193- 201011047 is different from the bHl-44 residue of bHl. The figure is modified from Figure 56; Figure 58 shows the binding of bHl-44 I29A Y32A bHl-44 and R50A R58A bHl-44 antibodies to VEGF and HER2. The ELISA binding assay demonstrated the ability of bHl-44 IgG and two double mutants to bind to biotinylated VEGF109 (left) or HER2-ECD (right) and to compete with immobilized anti-VEGF antibodies or Herceptin, respectively. The I29A/Y32A LC mutant, although maintaining similar affinity to bH1-44 for HER2, has lost VEGF binding ability. The R5 0A/R5 8A HC mutant has lost its affinity for HER2, but retains VEGF binding; Figure 59 shows the calorimetric measurement of the enthalpy change associated with antigen binding. Figure 59A-F shows binding of bH1 to VEGF, binding of bHl to HER2, binding of bHl-44 to VEGF, binding of bHl-44 to HER2, binding of bHl-44 HC-R50A + R58A to VEGF, bHl-, respectively. 44 LC-I29A + Y32A combined with HER2 data. The figures show individual heat pulses (top panel) and heat of reaction (bottom panel), which are calculated by integrating each pulse that has been plotted as a function of the ratio of antibody to antigen at the end of the injection. A small change in 需要 requires a relatively high protein concentration, which excludes an accurate estimate of KD when the affinity is high; Figure 59A-D: bHl or bHl-44 Fab at a concentration of 100 to 200 μΜ by 15 injections To titrate the VEGF109 or HER2-ECD solution at a concentration ranging from 10 to 20 μΜ. Figure 59E-F: titration of VEGF109 or HER2-ECD at a concentration of 10 to 20 μM by 20 injections of bHl-44 LC-I29A+Y32A Fab or bHl-44 HC-R50A+R58A Fab at a concentration of 150 and 250 μM Solution. Due to instrumental noise, the titrations of No. 1 and No. 13 of 142552.doc-194-201011047 were excluded from the analysis (Circle 59E); Figure 60 shows the thermodynamics of the bHl variant and the Herceptin® antibody. Each of the dual specific variants (bHl, bHl-81 and bHl-44) has a thermodynamic profile characterized by a favorable enthalpy and entropy for VEGF and HER2 binding. Variants HC-R50A+R58A and 1^-129 VIII + 丫3 2 VIII exhibiting loss of affinity for HER2 or VEGF, respectively, are shown in 1); a similar thermodynamic profile of «1-44. The thermodynamic profile of the 1^1-44/HER2 interaction is different from that of Herceptin/HER2; Figure 61 shows a comparison of bHl, bHl-44 and Herceptin® antibody hotspots for HER2 binding based on alanine scanning mutation-inducing data. Hot-spot residues are highlighted by gray located on the Herceptin® structure or the bHl Fab structure (bHl, bHl-44). A hotspot is defined as an AAG that is greater than or equal to 10% of the total combined free energy (AG). The structural contact sites (within 4.5 A of the antigen in the structure) are outlined in light dashed lines. HC and LC are separated by a black dotted line. The sequence of the underlined residue is different

Herceptin® ;

Figure 62 shows estimated heat capacity changes associated with binding of bHl-44 Fab to VEGF or HER2. The ACp is obtained from the slope of the temperature dependence of AH between 20 ° C and 37 ° C. Based on the linear relationship between AH and T', within this range, ACp appears to be independent of T (R = 0.919 ' for bHl-44/HER2 and R = 0.9989 for bHl-44/VEGF). The ACp of Herceptin®/HER2 was previously determined by Kelley et al. (Biochemistry' 1992); Figures 63A-B show the binding kinetics of the bHl-44 variant measured by BIAcore. These figures show the immobilization of (A) VEGF109 or (B) HER2-ECD with bHl-44 Fab (red), bHl-44-LC-Y32 (green), bHl-44-LC-142552.doc-195- 201011047 Overlap of a representative reaction versus time curve for the binding interaction between I29A+Y32A (magenta) and bHl-44-HC-R50A+R58A (grey) 0.5 μΜ solution. The traces indicate the combination with the same immobilized CM5 wafer, which is regenerated after each Fab operation. At 0.5 μΜ, no binding of bHl-44-LC-I29A+Y32A to VEGF or bHl-44-HC-R50A+R58A to HER2 was detected. The variant bHl-44-Y32A showed significantly weaker binding to VEGF compared to wild-type bHl-44; Figures 64A-D show that the specificity of bH1-44 determines the localization of the crystal structure based on bHl. Residues important for VEGF binding (LC-I29 and LC-Y32; A and B) and residues important for HER binding (HC-R50 and HC-R58; C and D) are shown in dark gray It is a rod on the crystal structure of bHl/VEGF (A and C, 2.6 A resolution) or bHl/HER2 (B and D, 2.9 A resolution). Residues 129 and Y32 appear to be involved in intrachain interactions to maintain the CDR-L1 loop configuration necessary for VEGF binding. 129 was exposed to the HER2 structure via solvent. Y32 overlaps with HER2 but is not involved in productive antigen contact. R50 and R58 overlap with D560 and E558 on HER2 and appear to be involved in charge-charge interactions. R50 and R5 8 were exposed to solvent in the VEGF solvent structure; and Figure 65 shows the performance of the Herceptin® mutant Fab (R50A, R58A and R50A/R58A). 142552.doc -196- 201011047 Sequence Listing &lt;110&gt;Multispecific Antibody &lt;120&gt;US-based Nandek Corporation&lt;130&gt; 50474/018002 &lt;140&gt; 098129415 &lt;141&gt; 2009-09-01 &lt;;150&gt; US 61/190,856 &lt;151&gt; 2008-09-03 &lt;160&gt; 85 &lt;170&gt; patentin version 3.5 &lt;210&gt; 1 &lt;211&gt;

&lt;212&gt; PRT &lt; 213 &gt; artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 1 Α5Π lie Ala Lys Thr lie Ser Gly Tyr 1 5 &lt;210&gt; 2 &lt;211&gt; 6 &lt;212&gt ; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide &lt;400&gt; 2 Trp Gly Ser Phe Leu Tyr

&lt;210&gt; 3 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

His Tyr Ser Ser Pro Pro 1 5 &lt;210&gt; 4 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence 142552 - Sequence Listing.doc 201011047 &lt;220&gt;&lt;223&gt;Synthetic peptide &lt;400&gt; 4

Asn lie Lys Asp Thr Tyr 1 5 &lt;210&gt; 5 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

Arg lie Tyr Pro Thr Asn Gly Tyr Thr Arg 15 10 &lt;210&gt; 6 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;22〗&gt; Synthetic peptide &lt;400&gt;

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 1 5 10 &lt;210&gt; 7 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

Asn lie Ser Gly Thr Tyr 1 5 &lt;210&gt; 8 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

Arg lie Tyr Pro Ser Glu Gly Tyr Thr Arg 1 5 10 -2- 142552 - Sequence Listing.doc 201011047 &lt;210&gt; 9 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt;Synthetic peptide &lt;400&gt; 9

Trp val Gly val Gly Phe Tyr Ala Met Asp 1 5 10 &lt;210&gt; 10 &lt;211&gt; 214 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly ·· asd Ara Val Thr lie Thr Cys Arg Ala ser Gin Asp val Asn Thr Ala 20 25 30 val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45

Tvr ser Ala Ser Phe Leu Tyr ser Gly val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Glv ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu asp Phe Ala Thr Tyr Tyr cys Gin Gin His Tyr Thr Thr Pro pro 85 90 95

Thr Phe Gly Gin Gly Thr Lys val Glu lie Lys Arg Thr val Ala Ala 100 105 110

Pro Ser val Phe lie Phe Pro pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125

Thr Ala ser Val val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 142552 - Sequence Listing.doc 201011047

Glu Ser Val Thr Glu Gin Asp ser 165

Ser Thr Leu Thr Leu ser Lys Ala 180

Ala cys Glu val Thr His Gin G]y 195 2〇〇

Phe Asn Arg Gly Glu Cys 210

Lys Asp Ser Thr Tyr Ser Leu Ser 170 175

Asp Tyr Glu Lys His Lys Val Tyr 185 190

Leu ser ser Pro val Thr Lys Ser 205 &lt;210&gt; 11 &lt;211&gt; 227 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;

Glu val Gin Leu val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 15 丄5 ser Leu Arg Leu ser Cys Ala Ala Ser Gly Phe Asn lie Lys Asp Thr ·&gt;λ jw

Tyr lie His Trp Val Arg Gin Ala Pro Gly Lys Gly Glu Trp val

Ala Arg lie Tyr Pro Thr Asn G&quot;ly Ding y "Thr Arg lyr Ala Asp Ser Val 50 55 60

Lys Gly Arq Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80

Leu Gin Met Asn ser Leu Arg Ala Glu Asp Thr Ala va! Tyr cys

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu val Thr Val ser ser Ala ser Thr Lys Gly Pro ser val 115 120 125

Phe Pro Leu Ala Pro Ser ser Lys ser Thr ser Gly Gly Thr Ala Ala 130 135 140

Leu Gly cys Leu Val Lys Asp Tyr Phe Pro Glu Pro val Thr val ser 145 150 155 160 142552 · Sequence Listing.doc -4 - 201011047

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala val 165 170 175

Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser ser val Val Thr Val Pro 180 185 190

Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys 195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser cys Asp 210 215 220

Lys Thr His 225

&lt;210&gt; 12 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

Asp val Asn Thr Ala val 1 5 &lt;210&gt;&lt;211&gt;&lt;212&gt;&lt;213&gt; 13 4 PRT artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 13 Ser Ala Ser Phe 1 &lt ;210&gt;&lt;211&gt;&lt;212&gt;&lt;213&gt; 14 4 PRT artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 14 His Tyr Thr Thr 1 &lt;210&gt; 15 142552 - Sequence Listing. Doc 201011047 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 15 Asn val 1 Trp Asp Trp val 1 5 &lt;210&gt; 16 &lt;211&gt 4 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 16 Pro Ala ser ser 1 &lt;210&gt; 17 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 17 Gly Trp Tyr lie Ala 1 5 &lt;210&gt; 18 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;;&lt;223&gt; Synthetic peptide &lt;400&gt; 18

Asp lie Pro Arg Ser He Ser Gly Tyr Val 1 5 10 &lt;210&gt; 19 &lt;211&gt; 4 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide-6 - 142552 - Preface List .doc 201011047

&lt;400&gt; 19 Trp Gly Ser Tyr 1 &lt;210&gt; 20 &lt;211&gt; 4 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 20 His Tyr Thr Thr 1 &lt;210&gt; 21 &lt;2U&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 21 Asp lie Gly Leu Gly Ser val 1 5 &lt;210&gt; 22 &lt;211&gt; 4 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 22 Trp Ala ser Tyr 1 &lt;210&gt; 23 &lt;211&gt; 7 &lt;212&gt ; PRT &lt; 213 &gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic peptide &lt;400&gt; 23 Asp lie Arg ser Gly Ser val &lt;210> 24 142552 - Sequence Listing.doc 201011047 &lt;210&gt; 25 &lt;211&gt; 12 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Sequence &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (4)..(5) &lt;22B&gt; n is a, c, g or t &lt;220&gt;&lt;221&gt; rm'sc_feature &lt;222&gt; (6)·.(6) &lt;223&gt; g and t exist in equal molar ratio &lt;;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; (7) · (8) &lt;223&gt; n is a, c, g or t &lt;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; ) (C9) &lt;223&gt; g and t exist in equal molar ratio &lt;400&gt; 25 catnnknnkr st &lt;210&gt; 26 &lt;211&gt; 12 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;;223&gt; Synthetic sequence &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (4)·(9) &lt;223&gt; a, g, c, and t exist in equal molar ratio &lt;400&gt; 26 kmtnnnnnnr st

12 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;22B&gt; Synthetic peptide &lt;400&gt; 24 Tyr Ser Thr val Pro Trp 1 5 142552 - Sequence Listing.doc 12 201011047 &lt; 210 &lt; 211 &gt; 12 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (4), (9) &lt;223&gt; a, g, c, and t exist in the same molar ratio 12 &lt;400&gt; 27 dggnnnnnnr st &lt;210&gt; 28 &lt;211&gt; 12 &lt;212&gt; DNA &lt;213&gt;

&lt;220&gt;&lt;223&gt; Synthetic sequence &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (1)..(2) &lt;223&gt; η is a, c, g or t &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (3)..(3) &lt;223&gt;g&gt;^t exists in equal molar ratio &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; 10)·(11) &lt;223&gt; η is a, c, gilt &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (12)·(12) &lt;223&gt; § and 1 to wait Mohr than 12 &lt;400&gt; 28 nnkgsttccn nk &lt;210&gt; 29 &lt;211&gt; 12 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence &lt;220&gt;&lt;221&gt; misc_feature 142552-Sequence table.doc 12 201011047 &lt;222&gt; (10)·(11) &lt;223&gt; η is a, c, g or t &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (12)..(12) &lt;223&gt; g and t exist in equal molar ratio &lt;400&gt; 29 tgggsttccn nk &lt;210&gt; 30 &lt;211&gt; 12 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt;&lt;223&gt; Synthetic sequence &lt;400&gt; 30 kgggsttcct mt &lt;210&gt; 31 &l t; 211 &gt; 12 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic sequence &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (1).. (2) &lt;223&gt; η is a, c, g or t &lt;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; (3)·. (3) &lt;223&gt; g and t are present in the ear-ear ratio&lt;400&gt; 31 nnkgsttcct mt &lt;210&gt; 32 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic sequence &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (1)..(1) &lt;223&gt;&lt;220&gt; g accounts for 70%, t accounts for 10%, a accounts for 10% and c accounts for 10% 12 12 142552 - Sequence Listing. doc 10-201011047 &lt;221&gt; Mi sc a feature &lt;222&gt; (2). (2) &lt;223&gt; gtt 10%, t is 10%, a is 70% and c is 10% &lt;220&gt;&lt;221&gt; mi sc a feature &lt ;222&gt; (3)..(3) &lt;223&gt; g accounted for 10%, t accounted for 10%, a accounted for 10% and c accounted for 70% &lt;220&gt;&lt;221&gt; misc^feature &lt;222&gt; 7)···(8) &lt;223&gt; n is a, c, g or t &lt;220&gt;&lt;221&gt; nri sc a feature &lt;222&gt; (9).(9) &lt;223&gt; Mobibi exists &lt;220&gt;

&lt;2 21&gt; misc_feature &lt;222&gt; CIO)..¢11) &lt;223&gt; n is a, c, g salty &lt;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; (12). .C12) &lt;223&gt; g and t exist in equal molar ratio &lt;400&gt; 32 nnnrttnnkn nktacsta &lt;210&gt; 33 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt; 223 &gt; Synthetic sequence &lt;220&gt;&lt;221&gt;&lt;222&gt;&lt;223&gt; mi sc a feature CD .. Cl) g accounted for 70%, t accounted for 10%, a accounted for 10% and c accounted for 10% &lt;;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (2)..(2) &lt;223&gt; 笆 10%, t accounted for 10%, a accounted for 70% and c accounted for 10% &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (3)..(3) &lt;223&gt; g accounts for 10%, t accounts for 10%, a accounts for 10% and c accounts for 70% &lt;220> -11 - 142552 - Sequence table.doc l&gt;&gt;&gt;&gt; 012 3 2222 2222 VVV &lt; 201011047 &lt;221&gt; misc^feature &lt;222&gt; ¢7)..(8) &lt;223&gt; n is a 'c, g or t &lt;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; (9) &quot;(9) &lt;223&gt; g and t exist in equal molar ratio &lt;220&gt;&lt;221&gt; a feature &lt;2 22&gt; (10)-.(11) &lt;223&gt; n is a, c, g or t &lt;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; (12)..(12) &lt;223&gt ; g and t are equal to the presence of &lt;400&gt; 33 nnnrttnnkn nkdggsta &lt;210&gt; 34 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence&lt;223&gt;;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (1)· (1) &lt;223&gt; g accounted for 70%, t accounted for 10%, a accounted for 10% and c accounted for 10% mi sc-feature (2)..(2) g accounts for 10%, t accounts for 10%, a accounts for 70% and c accounts for 10% &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (3)..(3) &lt;223&gt; g accounts for 10%, t 10%, a is 10% and c is 70% &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (7)..(8) &lt;223&gt; n is a, c, g or t &lt;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; (9)..(9) &lt;223&gt; g anti-t is present in the ear-earth ratio &lt;220&gt;&lt;221&gt; mi sc-feature &lt ;222&gt; (10)·(11)

142552· Sequence Listing.doc 12- 201011047 &lt;223&gt; η is a, c, g or t &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (12)..(12) &lt;223&gt; gAt exists in equal molar ratio &lt;220&gt;&lt;221&gt; mi sc-feature &lt;222&gt; (13)..(13) &lt;22B&gt; n is a, c, gilt &lt;400&gt; 34 nnnrttnnkn nknmtsta 18 &lt;210&gt; 35 &lt;211&gt; 4 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 35 val Asn hr Ala 1

&lt;210&gt; 36 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; B6 lie Pro Arg Ser lie Ser Gly Tyr 1 5 &lt;210&gt; 37 &lt;211&gt; 4 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 37

Ser Ala ser Phe 1 &lt;210&gt; 38 &lt;211&gt; 4 &lt;212&gt; PRT &lt;213&gt; artificial sequence-13 - U2552 - Sequence Listing.doc 201011047 &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 38

Trp Gly ser Tyr 1 &lt;210&gt; 39 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

"pp &lt;211&gt Tyr 1 &lt;210&gt; 41 &lt;211&gt; 6 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 41 His Tyr Thr Thr Pro Pro 1 5 &lt;210&gt; 42 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; artificial sequence

&lt;220&gt;&lt;223&gt;Synthesis&lt;400&gt; 42

Gin val Gin Leu val Gin Ser Gly Ala Glu val Lys Lys Pro Gly Ala 15 10 15 142552 - Sequence Listing.doc -14- 201011047

Ser val Lys val ser Cys Lys Ala ser Gly Thr Phe Thr 20 25 30 &lt;210&gt; 43 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;40 (h&gt;· 43

Trp val Arg Gin Ala pro Gly Gin Gly Leu Glu Trp Met Gly 1 5 10 &lt;210&gt; 44 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt;

&lt;220&gt;&lt;223&gt;Synthetic peptide &lt;400&gt; 44

Arcj val Thr1 lie Thr Ala Asp Thr Ser Thr ser Thr Ala Tyr Met Glu 1 5 l〇 15

Leu Ser Ser Leu Arg Se "Glu Asp Th" Ala Val Tyr Tyr Cys Ala Arg 20 25 30 &lt;210&gt; 45 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 45 T "p Gly Gin Gly Th" Leu val Th "val se "ser &lt;210&gt; 46 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt;Synthetic peptide &lt;400&gt; 46

Gin val Gin Leu val Gin ser Gly Ala Glu val Lys Lys Pro Gly Ala 142552 - Sequence Listing.doc -15- 201011047 10 15

Ser val Lys val ser Cys Lys Ala Ser 20 25 &lt;210&gt; 47 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic peptide &lt;400&gt;

Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met IS 10 &lt;210&gt; 48 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide &lt;400&gt; 48

Arg val Thr lie Thr Ala Asp Thr Ser Thr ser Thr Ala Tyr Met Glu 1 5 l〇 15

Leu Ser Ser Leu Arg ser Glu Asp Thr Ala Val Tyr Tyr cys Ala 20 25 BO &lt;210&gt; 49 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;;400&gt; 49

Arg val Thr lie Thr Ala Asp Thr Ser Thr ser Thr Ala Tyr Met Glu 1 5 l〇 15

Leu ser ser Leu Arg Ser Glu Asp Thr Ala val Tyr Tyr cvs 20 25 30 &lt;210&gt; 50 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt; 142552 - Sequence Listing.doc -16 - 201011047 &lt;223&gt;Synthetic peptide &lt;400&gt; 50

Gin val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys pro Ser Gin 15 10 15

Thr Leu ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser 20 25 30 &lt;210&gt; 51 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide&lt;400&gt; 51

Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie Gly 1 5 10 &lt;210&gt; 52 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;, &lt;223&gt; Synthetic peptide &lt;;400&gt; 52

Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe ser Leu Lys 15 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala val Tyr Tyr Cys Ala Arg 20 25 30 % &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide &lt;400&gt; 53

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu val Lys Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr val Ser 20 25 &lt;210&gt; 54 142552 - Sequence Listing.doc -17- 201011047 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 54

Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie 1 5 10 &lt;210&gt; 55 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic &lt;400&gt;; 55

Arg Vat Thr lie Ser val Asp Thr ser Lys Asn Gin Phe Ser Leu Lys 15 10 15

Leu Ser Ser val Thr Ala Ala Asp Thr Ala val Tyr Tyr Cys Ala 20 25 30 &lt;210&gt; 56 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide&lt;223&gt;;400&gt; 56

Arg val Thr lie ser val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys 15 10 15

Leu Ser ser val Thr Ala Ala Asp Thr Ala val Tyr Tyr cys 20 25 BO &lt;210&gt; 57 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 57

Glu val Gin Leu val Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 15 10 15 142552 - Sequence Listing.doc -18- 201011047 se, Leu ΛΓ9, eU se, Cvs Ala A1a Sf ^ -e Th. PHe Ser &lt;210&gt; 58 &lt;211&gt; 14

&lt;212&gt; PRT &lt;213&gt; artificial sequence · &lt;220&gt;&lt;223&gt; synthetic peptide &lt;400&gt;

Trp val Arg Gin Ala Pro Gly Lys Gly Glu Trp val Ser &lt;210&gt; 59 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt;

&lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 59

Arg Phe Thr lie Ser Arg Asp Asn Ser L^s Asn Thr Leu Tyr Leu Gin

Cys Ala Arg

Met Asn ser Leu Arg Ala du Asp Thr A!a VaT Tyr Tyr &lt;210&gt; 60 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 60

Glu val Gin Leu val Glu ser Gly Gly Gly Leu val Gin Pro Gly Gly ser Leu Arg Leu Ser Cys Ala Ala ser 20 25 &lt;210&gt; 61 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; Artificial sequence&lt;220&gt;&lt;223&gt;Synthetic peptide &lt;400&gt; 61 142552 - Sequence Listing.doc -19- 201011047

Trp val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp val 1 5 10 &lt;210&gt; 62 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt;artificial sequence&lt;220&gt;&lt;223&gt;Synthesis&lt;400&gt;; 62

Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu f;in 1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr cys Ala 20 25 B〇&lt;210&gt; 63 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide &lt;400&gt; 63

Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin 15 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 &lt;210&gt; 64 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide&lt;400&gt; 64

Glu val Gin Leu Val Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 15 10 15 ser Leu Arg Leu Ser cys Ala Ala ser Gly Phe Asn lie Lys 20 25 30 &lt;210&gt; 65 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial sequence 142552 - Sequence Listing. doc -20- 201011047 &lt;220&gt;&lt;223&gt;Synthetic peptide &lt;400&gt; 65

Arg Phe Thr lie Ser Ala Asp Thr Ser lys Asn Thr· Ala Tyr Leu Gin 1 5 l〇 15

Met Asn ser # Arg Ala Glu asp # A vd Tyr Tyr want ser Arg &lt;210&gt; 66 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic peptide &lt;400&gt;; 66

Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gin 1 5 l〇 is

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Typ Tyr Cys Ser 20 25 3〇&lt;210&gt; 67 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; Peptide &lt;400&gt; 67 15

Arg Phe Thr lie !e"Ala Asp Thr Ser s Asr&gt; Thr Ala Tyr Leu Gin

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr 20 25

Cys Ala Arg &lt;210&gt; 68 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic peptide &lt;400&gt;

Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gin 15 10 15 142552 - Sequence Listing.doc •21- 201011047

ET Ala Glu Asp Thr Ala val Tyr Tyr cvs Ala 20 25 30 &lt;210&gt;;400&gt; 69

Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gin 15 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala val Tyr Tyr Cys 20 25 30 &lt;210&gt; 70

&lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 70

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala ser val Gly 15 10 15

Asp Arg val Thr lie Thr Cys 20 &lt;210&gt; 71 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt;

&lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 71

TrD Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr 1 5 10 15 &lt;210&gt; 72 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; 142552-Sequence List.doc -22- 201011047 &lt;400&gt; 72

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15

Leu Thr lie ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr cys 20 25 30 &lt;210&gt; 73 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt;&lt;400&gt; 73

Phe Gly Gin Gly Thr Lys Val Glu lie Lys 15 10

&lt;210&gt; 74 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic peptide &lt;400&gt; 74

Asp lie val Met Thr Gin ser Pro Leu Ser Leu Pro val Thr pro Gly 15 10 15

Glu Pro Ala Ser lie Ser Cys 20 &lt;210&gt; 75 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu lie Tyr 15 10 15 &lt;210&gt; 76 &lt;211&gt; B2 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide 142552 - Sequence Listing.doc -23- 201011047 &lt;400&gt; 76

Gly val Pro Asp Arg Phe ser Gly ser Gly Ser Gly Thr Asp phe Thr 1 5 10 15

Leu Lys lie ser Arg val Glu Ala Glu Asp val Gly val Tyr Tyr cys 20 25 30 &lt;210&gt; 77 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt;&lt;400&gt; 77

Glu lie val Leu Thr Gin Ser Pro Gly Thr Leu ser Leu Ser pro Gly 15 10 15

Glu Arg Ala Thr Leu ser Cys 20 &lt;210&gt; 78 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt;

Trp Tyr Gin Gin Lys Pro Gly Gin AT a Pro Arg Leu Leu lie Tyr &lt;210&gt; 79 &lt;211&gt; 32

&lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic peptide &lt;400&gt;

Gly He Pro Asp Arg Phe ser Gly Gly sen Gly Thr Asp Phe Thr , i , . . m.. Glu Asp Phe Ala val Tyr Tyr cys

Leu Thr lie Ser Arg Leu Glu Pr° a〇J 7 20 mouth &lt;210&gt; 80 &lt;211&gt; 23 142552 · Sequence Listing.doc 201011047 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic peptide &lt;400&gt; 80 Asp lie Val Met Thr Gin Ser pro Asp Ser Leu Ala Val Ser Leu Gly 15 10 15

Glu Arg Ala Thr lie Asn Cys 20 m &lt;210&gt; 81 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Construct &lt;400&gt; 81 Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Tyt 15 10 15 &lt;210&gt; 82 &lt;211&gt; 32 &lt;212&gt; PRT &lt;2lJ&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthetic Peptide &lt;400&gt; Gly val Pro Asp Arg Phe Ser Gly Ser Gly ser Gly Thr Asp Phe Thr 15 10 15 Leu Thr He Ser ser Leu Gin Ala Glu Asp Val Ala val Tyr Tyr Cys 20 25 30 &lt;210&gt; 83 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence ^^Λ &gt; 2222 2222 VVV &lt; MISC_FEATURE(1)..(1) Xaa is any amino acid other than Asp &lt;;220&gt; 142552-Sequence List.doc 25- 201011047 &lt;221&gt; MISC_FEATURE &lt;222&gt; (3)..(3) &lt;223&gt; Xaa is any amino acid other than Pro &lt;220&gt;&lt;221&gt;; MISCL.FEATURE &lt;222&gt; (4)..(4) &lt;223&gt; Xaa is any amino acid other than Arg &lt;220&gt;&lt;221&gt; MISC_FEATURE &lt;222&gt; (5). ) &Lt;223&gt; Xaa is any amino acid other than Ser &lt;220&gt;&lt;221&gt; MISC_FEATURE &lt;222&gt; (6)··C9) &lt;223&gt; Xaa is any amino acid &lt;400&gt; 83 xaa Lie Xaa xaa Xaa xaa xaa xaa xaa Tyr 15 10 &lt;210&gt; 84 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic sequence &lt;220&gt;&lt;221&gt;; MISC_FEATURE &lt;222&gt; (2)..(9) &lt;223&gt; Xaa is any amino acid &lt;400&gt; 84

Arg xaa xaa Xaa xaa Xaa xaa Xaa xaa Arg 15 10 &lt;210&gt; 85 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthetic sequence &lt;220&gt;;221&gt; MISC-FEATURE &lt;222&gt; (2)..(4) &lt;22B&gt; Xaa is any amino acid &lt;220&gt;&lt;221&gt; MISC_FEATURE &lt;222&gt; (5)·(5) &lt;;223&gt; Xaa is any amino acid other than Thr 26- 142552 - Sequence Listing. doc 201011047 &lt;220&gt;&lt;22X&gt; MISCUFEATURE &lt;222&gt; (6)..(6) &lt;223&gt; Xaa is a system Any amino acid other than Asn &lt;220&gt;&lt;221&gt; MISC_FEATURE &lt;222&gt; (7)..(9) &lt;223&gt; Xaa is any amino acid &lt;400&gt;

Arg Xaa Xaa Xaa xaa xaa xaa Xaa xaa Arg 15 10

-27- 142552 - Sequence Listing.doc

Claims (1)

201011047 VII. Patent Application Range: 1. An isolated antibody comprising a hypervariable region (HVR) L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1), wherein the antibody specifically binds to human epidermal growth Factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF). 2. The antibody of claim 1, wherein the antibody further comprises one or two HVR sequences selected from the group consisting of: (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); and (9) comprising the sequence HVR_L3 of HYSSPP (SEQ ID NO: 3). 3. The antibody of claim 1, wherein the antibody further comprises one, two or three HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the sequence NIKDTY (SEQ ID NO: 4); Ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and (iii) HVR-H3 comprising the sequence WGGDGF YAMD (SEQ ID NO: 6). 4. The antibody of claim 1, wherein the antibody further comprises one, two or three H VR sequences selected from the group consisting of: (i) HVR-H1 comprising the sequence NISGTY (SEQ ID ΝΟ·7); (ϋ) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) comprising the sequence WVGVGFYAMD (SEQ ID NO: 9) HVR-H3. 5. The antibody of claim 1, wherein the antibody binds human and murine VEGF with a Kd of 150 nM or greater and binds HER2 with a Kd of 7 nM or greater. 6. The antibody of claim 1, wherein the antibody inhibits VEGF-induced cell proliferation and HER2-expressing cell proliferation relative to a control. 7. The antibody of claim 1, wherein the antibody inhibits binding of VEGF to VEGF receptor 142552.doc 201011047 2 (VEGFR2). 8. An isolated antibody, wherein the antibody binds to human and murine VEGF with a Kd of 150 nM or stronger and binds to HER2 with a Kd of 7 nM or greater, and wherein the antibody inhibits VEGF-induced cells relative to the control Proliferation and HER2 express the proliferation of cells. 9. The antibody of claim 8, wherein the antibody binds to human and murine VEGF with a Kd of 36 nM or greater and binds to HER2 with a Kd of 1 nM or greater. 10. An isolated antibody fragment, wherein the anti-purine fragment binds to human VEGF with a Kd of 58 nM or stronger and binds to HER2 with a Kd of 6 nM or greater, and wherein the anti-purine fragment inhibits VEGF induction relative to the control The cells are thinned and HER2 shows the proliferation of cells. 11. The antibody fragment of claim 10, wherein the antibody fragment binds human and murine VEGF with a Kd of 33 nM or greater and binds HER2 with a Kd of 0.7 nM or greater. 12. The antibody fragment of claim 10 or 11, wherein the fragment is a Fab fragment. 13. An isolated antibody, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each sequence comprises SEQ ID NO: 1, 2, 3 Sequences of 4, 5 and 6, and wherein the antibody specifically binds to HER2 and VEGF. 14. An isolated antibody, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each sequence comprises SEQ ID NO: 1, 2, 3, Sequences 7, 8, and 9, and wherein the antibody specifically binds to HER2 and VEGF. An isolated antibody comprising an HVR L1 sequence, the HVR L1 sequence 142552.doc 201011047 comprising the sequence XdXsX.XsXsAXsXpYCSEQ ID NO: 83), wherein X! is any amino acid other than Asp, and X3 is in addition to Pro Any amino acid other than X4 is any amino acid other than Arg, and χ5 is any amino acid other than Ser, and wherein the antibody specifically binds to HER2 and VEGF. 16. The isolated antibody of claim 15, wherein Χι is Asn, X3 is Ala, X4 is Lys, X5 is Thr, or any combination thereof. 17. The isolated antibody of claim 15 or 16, wherein χ5 is Thr, X7 is Ser and X8 is Gly. 18. The isolated antibody of claim 15, wherein the antibody further comprises one or two HVR sequences selected from the group consisting of: (1) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); and (ii) The HVR-L3 comprising the sequence HYSSPP (SEQ ID NO: 3). The antibody according to claim 15, wherein the antibody further comprises one, two or three HVR sequences selected from the group consisting of: (1) inclusion HVR-H1 of the sequence NIKDTY (SEQ ID NO: 4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6) . 20. The isolated antibody of claim 15, wherein the antibody further comprises one, two or three HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the sequence NISGTY (SEQ ID NO: 7) (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). An antibody according to claim 15, wherein the antibody binds to human and murine VEGF with a Kd of 150 nM or more and binds with Kd of 7 nM or more 142552.doc 201011047 HER2. 22. The isolated antibody of claim 15, wherein the antibody inhibits VEGF-induced cell proliferation and HER2 expressing cell proliferation relative to a control. 23. The isolated antibody of claim 15, wherein the antibody inhibits binding of VEGF to VEGFR2. 24. The isolated antibody of claim 15, wherein the antibody further comprises an HVR H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 84), and wherein the antibody specifically binds to HER2 and VEGF. 25. An isolated antibody comprising an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein X5 is any amino acid other than Thr and X6 is any amino acid other than Asn And wherein the antibody specifically binds to HER2 and VEGF. 26. The isolated antibody of claim 25, wherein X5 is Ser, X6 is Glu, or X5 is Ser and X6 is Glu. 27. An isolated antibody according to claim 25 or 26, wherein the sense 8 is 7丫1·. 28. The isolated antibody of claim 25, wherein the antibody further comprises one or two HVR sequences selected from the group consisting of: (1) HVR-H1 comprising the sequence NISGTY (SEQ ID NO: 7); (ii) comprising HVR-H3 of the sequence WVGVGFYAMD (SEQ ID NO: 9). The isolated antibody of claim 25, wherein the antibody further comprises one, two or three HVR sequences selected from the group consisting of: (1) HVR-L1 comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1); Ii) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2); (iii) HVR-L3 comprising the sequence HYSSPP (SEQ ID NO: 3). 30. The isolated antibody of claim 25, wherein the antibody binds to human and murine VEGF with a Kd of 150 nM or greater and binds HER2 with a Kd of 7 nM or greater. 3. The isolated antibody of claim 25, wherein the antibody inhibits VEGF-induced cell proliferation and HER2 expressing cell proliferation relative to a control. 32. The isolated antibody of claim 25, wherein the antibody inhibits binding of VEGF to VEGFR2. The antibody of any one of claims 1 to 9, 13 to 16, 25 and 26, wherein the antibody is a monoclonal antibody. The antibody of any one of claims 1 to 9, 13 to 16, 25 and 26, wherein the antibody is an IgG antibody. 35. A fragment of an antibody according to any one of claims 1 to 9, 13 to 16, 25 and 26, wherein the fragment specifically binds to HER2 and VEGF. 36. The fragment of claim 35, wherein the fragment is a Fab fragment or a single-chain variable fragment (scFv). The antibody or antibody fragment of any one of claims 1 to 9, 13 to 16, 25 and 26, wherein at least a portion of its framework sequence is a human consensus framework sequence 0 3 8. The antibody or antibody fragment of any one of claims 1 to 37. 39. A polynucleotide encoding an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1). a polynucleotide encoding an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1) and (i) an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID 142552.doc 201011047 NO: 2) or (ii) an HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO: 3) or both (i) and (ii). 41. The polynucleotide of claim 39 or 40, wherein the polynucleotide additionally encodes one, two or three HVR sequences selected from the group consisting of: (1) comprising the sequence NIKDTY (SEQ ID NO: 4) HVR-H1; (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6). 42. The polynucleotide of claim 39 or 40, wherein the polynucleotide additionally encodes one, two or three HVR sequences selected from the group consisting of: ©3 (i) comprising the sequence NISGTY (SEQ ID NO: 7) HVR-H1; (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). 43. A polynucleotide encoding an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7). 44. A polynucleotide encoding an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8). 45. A polynucleotide encoding an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). 46. A polynucleotide encoding an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7); an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and a sequence comprising the WVGVGFYAMD (SEQ ID NO: 9) HVR-H3 sequence. 47. A polynucleotide encoding the HVR-L1 sequence comprising the sequence XJX3X4X5X6X7X8X9Y (SEQ ID NO: 83), wherein X丨 is 142552.doc 201011047 in addition to Asp, any amino acid, X3 is other than Pro Any amino acid, X4 is any amino acid other than Arg, and X5 is any amino acid other than Ser. 48. A polynucleotide encoding the HVR-L1 sequence comprising the sequence XJXsXjXsXeXAsXpY (SEQ ID NO: 83) (where Xi is any amino acid other than Asp, and X3 is any amino acid other than Pro, X4 is any amino acid other than Arg, X5 is any amino acid other than Ser) and (i) HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO: 2) or (ii) comprises the sequence HYSSPP (SEQ ID N0: 3) HVR-L3 sequence or (i) and (ii) two miscellaneous. 49. The polynucleotide of claim 47 or 48, wherein X! is Asn, X3 is Ala, X4 is Lys' X5 is Thr' X7 is Ser' Xg is Gly or any combination thereof. 50. A polynucleotide encoding the HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein X5 is any amino acid other than Thr and X6 is any amino acid other than Asn . 51. A polynucleotide encoding an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7); an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NOJ5), wherein X5 is any amine group other than Thr The acid and X6 are any amino acids other than Asn; and the HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). 52. The polynucleotide of claim 50 or 51, wherein X5 is Ser, X6 is Glu, and X8 is Tyr or any combination thereof. 53. A polypeptide comprising an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID ΝΟ: 1). 54. A polypeptide comprising an HVR-L1 sequence comprising 142552.doc 201011047 column X〗 IX3X4X5X6X7X8X9Y (SEQ ID NO: 83) ' wherein X is any amino acid other than Asp, X3 In the case of any amino acid other than Pro, X4 is any amino acid other than Arg, and X5 is any amino acid other than Ser. 55. A polypeptide comprising: an HVR-L1 sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1) and (i) an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO: 2) or (ii) comprising the sequence HYSSPP The HVR-L3 sequence of (SEQ ID NO: 3) or both (i) and (ii). 56. A polypeptide comprising: an HVR-L1 sequence comprising the sequence XJX3X4X5X6X7X8X9Y (SEQ ID ΝΟ83) (wherein 丨 is any amino acid other than Asp, and X3 is any amino acid other than Pro, X4 is any amino acid other than Arg, X5 is any amino acid other than Ser) and (1) HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO: 2) or (ii) contains the sequence H4HYSSPP (SEQ ID NO: 3) HVR-L3 sequence 歹 4 or both (i) and (ii). 57. The polypeptide of claim 54 or 56, wherein 1 is Asn, X3 is Ala, X4 is Lys, X5 is Thr, X7 is Ser, and X8 is Gly or any combination thereof. 5. The polypeptide of claim 54, 55 or 56, wherein the polypeptide further comprises one, two or three HVR sequences selected from the group consisting of: (1) HVR comprising the sequence NIKDTY (SEQ ID NO: 4) H1; (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID ΝΟ··5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6). 5. The polypeptide of claim 54, 55 or 56, wherein the polypeptide further comprises one, two or three HVR sequences selected from the group consisting of: 142552.doc • 8 - 201011047 (i) comprising the sequence NISGTY ( HVR-H1 of SEQ ID NO: 7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). 60. A polypeptide comprising an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7). 61. A polypeptide comprising an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8). 62. A polypeptide comprising an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). 63. A polypeptide comprising an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein X5 is any amino acid other than Thr and X6 is any amino acid other than Asn. 64. A polypeptide comprising: an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7); an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and a sequence comprising the WVGVGFYAMD (SEQ ID NO: 9) %&gt;^HVR-H3 sequence. 65. A polypeptide comprising: an HVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO: 7); and an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein X5 is any amine other than Thr The base acid and X6 are any amino acid other than Asn; and the HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9). 66. The polypeptide of claim 63 or 65, wherein X5 is Ser, X6 is Glu, and X8 is Tyr or any combination thereof. 67. A vector comprising a polynucleoside 142552.doc -9- 201011047 acid as claimed in any one of claims 38 to 52. 68. 70. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. January = the carrier of item 67 where the vector is the expression vector. = host cell comprising the vector of claim 67 or 68. : The host cell of claim 69 wherein the host cell is a prokaryotic cell. The host cell of ^ 69 is the eukaryotic cell. The host cell of claim 71, wherein the host cell is a mammalian cell. The method of producing an antibody or antibody fragment according to any one of claims 1 to 37, comprising culturing a host cell comprising a vector comprising the polynucleotide of any one of claims 38 to 52 and recovering the antibody. The method of claim 73 wherein the host cell is a prokaryotic cell. The method of claim 73, wherein the host cell is a eukaryotic cell. The method of claim 75, wherein the host cell is a mammalian cell. Use of an antibody or antibody fragment according to any one of claims 37 to 37 for the manufacture of a medicament for treating a tumor of an individual, wherein the administration time and administration amount of the medicament is sufficient to treat or prevent the individual The tumor. For example, the use of the item 77, wherein the tumor is a colorectal tumor, a breast cancer lung cancer, a renal cell carcinoma, a glioma, a glioblastoma or an ovarian cancer. The use of claim 77, wherein the drug is administered with another anti-cancer therapy. The use of claim 79, wherein the another anticancer therapy comprises another antibody or antibody fragment, a chemotherapeutic agent, a cytotoxic agent, an anti-angiogenic agent, 142552.doc 201011047, an inhibitor, a rigid drug, a cytokine (cytokine) , cytokine antagonists, cytotoxic radiation therapy, corticosteroids, antiemetics, cancer vaccines, analgesics or growth inhibitors. 81. The use of claim 79, wherein the another anti-cancer therapy is administered before or after administration of the drug. 82 is the use of the term 79, wherein the other anti-cancer therapy is administered concurrently with the drug. 83. The antibody or antibody fragment according to any one of claims 1 to 37, which is used for the manufacture of a medicament for treating an autoimmune disease in an individual, wherein the administration time of the drug And administering the autoimmune disease in an amount sufficient to treat or prevent the individual. 84. Use of an antibody or antibody fragment according to any one of claims 1 to 37 for the manufacture of a medicament for treating a non-malignant disease involving aberrant activation of HER2 in an individual, wherein the administration time and administration of the drug And the amount is sufficient to treat or prevent the non-malignant disease of the individual. % 85. The use of claim 77, 83 or 84 wherein the individual is a human. 142552.doc